Uc Davis/Nih Neuromab Facility: Immunofluorescence Staining of Free Floating Brain Sections

UC Davis/NIH NeuroMab Facility
Department of Neurobiology, Physiology and Behavior, UC Davis, Davis CA 95616-8519

www.neuromab.org neuromab@ucdavis.edu (530) 752-8398
Immunofluorescence Staining of Free Floating Brain Sections
Day 1.
1) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold 0.1 M PB (0.1M Na
Phosphate pH 7.4).
2) Block sections in vehicle (10% normal goat serum in 0.1 M PB+ 0.3% Triton X-100) for 1 h with
gentle rocking/agitation at 4ºC
3) Incubate sections in NeuroMab diluted in ice-cold vehicle. Dilution factors for NeuroMabs should
be determined empirically, but generally NeuroMab tissue culture supernatants should be used at
1:2-1:10, purified NeuroMab IgG from 1-20 µg/ml. Incubate sections overnight at 4ºC with gentle
rocking/agitation.
Day 2.
4) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold vehicle.
5) Incubate sections for 1 h with gentle rocking/agitation at 4ºC in appropriate fluorescent goat anti-
mouse secondary antibody. We typically use Alexa Fluor conjugated goat anti-mouse isotype
specific (i.e., anti-IgG1, IgG2a or IgG2b) secondary antibodies, which have a higher signal and
lower background than anti-IgG secondary antibodies.
From this step on the sections should be protected from room light.
6) Wash sections with gentle rocking/agitation at 4ºC, 5 min in vehicle.
7) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.1 M PB.
8) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.05 M PB.
9) Mount on gelatin-coated microscope slides (floating in 0.05 M PB in a 15 ml Petri dish).
10) Air dry
11) Put 15 µl mounting media (e.g., Prolong Gold antifade reagent, Invitrogen P36930) on each
section and coverslip.
12) Seal with nail polish.
A cooperative venture among the University of California at Davis, the National Institutes of Health, and Antibodies Inc.
http://neuromab.ucdavis.edu neuromab@ucdavis.edu (530) 752-8398
Immunofluorescence Labeling of Free-Floating Perfusion-Fixed Brain Sections
Note: protocol can be completed in one day by shortening primary antibody incubation (Step 3).
Day 1.
1) Section preparation: wash sections four times with ice-cold (4oC) 0.1 M PB (recipe below) with
gentle rocking/agitation at room temperature (RT) for 5 min each.
2) Block: incubate sections in vehicle (recipe below) with gentle rocking/agitation at 4°C for 1 h.
3) Primary antibody incubation: transfer sections into ice-cold (4oC) vehicle containing diluted
NeuroMab(s) and incubate with gentle rocking/agitation at RT for 3 h (or at 4°C overnight).
Note: antibody concentrations and incubation conditions should be determined empirically for each
combination of target sample, NeuroMab, secondary antibody and detection system but, as a
general guide, NeuroMabs should be used between 0.1 and 10 μg/mL.
Day 2.
4) Wash: transfer sections into fresh ice-cold (4oC) vehicle and wash a total of four times with
gentle rocking/agitation at RT for 5 min each.
5) Secondary antibody incubation: transfer sections into ice-cold (4oC) vehicle containing
fluorescently-conjugated anti-mouse secondary antibodies diluted as per manufacturer’s
recommendations (e.g., 0.5 μg/mL of Alexa Fluor 488 conjugated anti-mouse IgG1 antibody,
Thermo Fisher Scientific catalog # A-21121) and incubate with gentle rocking/agitation protected
from light at 4°C for 1 h.
Note: use subclass-specific secondary antibodies matched to the NeuroMab IgG subclass (i.e.,
IgG1, IgG2a or IgG2b) for optimal signal detection and lowest background (Manning et al., PLoS
One 7:e38313, 2012, http://www.ncbi.nlm.nih.gov/pubmed/22675541).
6) Wash: transfer sections into 0.1 M PB, wash twice with gentle rocking/agitation at RT for 5 min
each and wash once with 0.05 M PB with gentle rocking/agitation at RT for 5 min.
7) Mounting: transfer sections into 15 mL Petri dish containing 0.05 M PB, carefully mount onto
gelatin-coated microscope slides and allow to air dry.
8) Sudan Black B treatment (optional, see below).
9) Coverslipping: pipet anti-fade medium (e.g., ProLong Gold Antifade Mountant, Thermo Fisher
Scientific catalog # P36930) on each section according to manufacturer’s instructions, place
A cooperative venture between the University of California at Davis, the National Institutes of Health and Antibodies Incorporated
last updated 14Oct2016 page 1 of 2

glass coverslip on top, cure for at least 24 hours and seal edges with nail polish.
Note: tissue autofluorescence can be reduced with an optional Sudan Black B treatment (SBB,
Oliveira et al., Histol Histopathol 25:1017, 2010, https://www.ncbi.nlm.nih.gov/pubmed/20552552).
1) Completely rehydrate slide-mounted sections with ddH20
2) Completely submerge slide in a tray containing 0.05% SBB (recipe below).
3) Incubate with gentle rocking/agitation at RT for 5 min. Sections will become blue.
4) Remove slide from tray and rinse well with ddH20, paying attention to remove any SBB
particulates.
5) Air dry and proceed to coverslipping step.
Recipes:
0.1 M PB: Dilute 500 mL of stock 0.4 M PB (see perfusion protocol for recipe) into ≈1400 mL
ddH20, adjust pH to 7.4 and bring to final volume of 2 L.
Vehicle: 10% normal goat serum and 0.3% Triton X-100 in 0.1 M PB
SBB solution:
1) Prepare stock solution of 1% SBB (Electron Microscopy Sciences catalog # 21610) in 100%
ethanol.
2) Vortex or place into a sonicating water batch for 1 h.
3) Vacuum filter SBB through 0.45 μM cellulose acetate, low protein binding membrane
(Corning catalog # 430314).
4) Prepare working 0.05% SBB solution. Dilute 2.5 mL SBB stock in 47.5 mL 70% ethanol
immediately prior to each use in order to minimize particulates. If autofluorescence levels
are high, then higher SBB concentrations (up to 0.15%) may be justified but increasing SBB
concentrations may also reduce immunofluorescence signal.
Note: stock SBB solution can be reused but must be sonicated for at least 1 h prior to preparing
working solution.
Refs: Oliveira et al., Histol Histopathol 25:1017, 2010 (https://www.ncbi.nlm.nih.gov/pubmed/20552552);

Manning et al., PLoS One 7:e38313, 2012 (https://www.ncbi.nlm.nih.gov/pubmed/22675541);
Bishop et al., J Neurosci 35:14922, 2015 (https://www.ncbi.nlm.nih.gov/pubmed/26538660)

www.neuromab.org neuromab@ucdavis.edu (530) 752-8398
Antigen Retrieval Methods for Free Floating Brain Sections
1) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold 0.1 M PB (0.1M Na
Phosphate pH 7.4).
2) Antigen retrieval
Option 1: Citrate method: Wash sections with 10 mM sodium citrate, pH 8.5 for 5 min at room temperature
with gentle agitation. Incubate sections in 10 mM sodium citrate, pH 8.5 for 30 min at 80° C with gentle
agitation.
Option 2. Pepsin method: Wash sections in purified H2O for 5 min at 37° C with gentle agitation. Incubate
sections in 0.15 mg/ml pepsin dissolved in 0.2N HCl for 10 min at 37° C with gentle agitation.
3) Cool sections to RT
4) Wash sections 2X in cold 0.1M PB
5) Proceed to the blocking step on either the immunoperoxidase (step 4) or immunofluorescence (step 2)
staining protocol.
References:
Citrate: Jiao et al., J Neurosci Methods 93:149, 1999
Pepsin: Fukaya & Watanabe, J Comp Neurol 426:572, 2000.
A cooperative venture among the University of California at Davis, the National Institutes of Health, and Antibodies Inc.
Immunoperoxidase Labeling of Free-Floating Perfusion-Fixed Brain Sections
Day 1.
For antibody screening, prior to the primary antibody step, all incubation steps can be accelerated
using 6-well plates with net inserts containing 10-15 sections in each well.
1) Wash: transfer sections to 6-well inserts and rinse twice with ice-cold (4oC) TBS (recipe below)
with gentle rocking/agitation at room temperature (RT) for 5 min each.
2) Permeabilization: transfer sections into ice-cold (4oC) TBS containing 0.5% Triton X-100 and 10
μg/mL Avidin (egg white avidin, Thermo Fisher Scientific catalog # A887) with gentle
rocking/agitation at 4°C for 45 min.
3) Wash: transfer sections into fresh ice-cold (4oC) TBS and wash twice with gentle
rocking/agitation at RT for 5 min each.
4) Block: incubate sections in ice-cold (4oC) vehicle (recipe below) containing 18.75 μg/mL Biotin
(Sigma-Aldrich catalog # B4501-1G) with gentle rocking/agitation at 4°C for 45 min.
5) Primary antibody incubation: transfer sections into ice-cold (4oC) vehicle containing diluted
NeuroMab(s) and incubate with gentle rocking/agitation at 4°C overnight.
Note: antibody concentrations and incubation conditions should be determined empirically for each
combination of target sample, NeuroMab, secondary antibody and detection system but, as a
general guide, NeuroMabs should be used between 0.1 and 10 μg/mL.
Day 2.
6) Wash: transfer sections into ice-cold (4oC) TBS and wash a total of five times with gentle
7) Secondary antibody incubation: transfer sections into ice-cold (4oC) vehicle containing
biotinylated anti-mouse antibodies diluted as per manufacturer’s recommendations (e.g., 0.5
μg/mL of biotinylated horse anti-mouse IgG H+L antibody, Vector Labs catalog # BA-2001) and
incubate with gentle rocking/agitation at 4°C for 1 h. Alternatively, a cocktail of subclass-specific
secondary antibodies matched to the NeuroMab IgG subclass (i.e., IgG1, IgG2a or IgG2b at 0.5
μg/mL each) can be used for optimal signal detection and lowest background (Manning et al.,
PLoS One 7:e38313, 2012, http://www.ncbi.nlm.nih.gov/pubmed/22675541).
8) ABC preparation: immediately before next round of washes, make ABC reagent (Vectastain Elite
Peroxidase System, Vector Labs catalog # PK-6100) according to manufacturer’s instructions
(i.e., a 1:1 ratio of Solution A and Solution B mixed and incubated at 4°C for 30 min).

9) Wash: transfer sections into ice-cold (4oC) TBS and wash a total of five times with gentle
10) ABC incubation: transfer sections into ice-cold (4oC) ABC solution and incubate with gentle
rocking/agitation at 4°C for 1 h.
11) Wash: transfer sections into ice-cold (4oC) TBS and wash a total of three times with gentle
12) Wash: transfer sections into ice-cold (4oC) TB (recipe below), wash twice with gentle
rocking/agitation at RT for 5 min each. During washes, prepare DAB/NAS solution (recipe
below).
13) DAB/NAS development: transfer sections into DAB/NAS solution and develop, taking care to
visually monitor the intensity of the reaction so as to avoid over-developing. To stop reaction,
transfer sections to TB and wash twice more with TB at RT.
14) Mounting: transfer sections into a 15 mL Petri dish containing TB, carefully mount onto gelatin-
coated microscope slides and dry using a slide warming tray (Fisher Scientific) for 20-30 min.
15) Dehydration: transfer slides with sections through 70%, 95% and 100% ethanol (twice), followed
by Richard-Allan Citrus Clearing Solvent (Thermo Fisher Scientific catalog # 8301), for 10 min
each in a fume hood.
16) Coverslipping: pipet DPX mountant (Electron Microscopy Sciences catalog # 13510) on each
slide according to manufacturer’s instructions, place glass coverslip on top and lay flat to set in
fume hood overnight.
Recipes:
TB: 50 mM Tris pH 7.5
TBS: 150 mM NaCl in 50 mM Tris pH 7.5
Vehicle: 5% normal horse/goat serum (secondary antibody host) and 0.1% Triton X-100 in TBS
DAB/NAS solution (100 mL, prepare immediately before use):

1) Dissolve 0.04 g DAB (3-3’ diaminobenzidine tetrahydrochloride) and 0.3 g NAS (nickel
ammonium sulfate) in 100 mL of TB and filter through a Whatman #2 filter.
2) Immediately before developing, add 10 µL of 30% H2O2.
3) Note that DAB is a suspected carcinogen. Wear protective gear and work under a hood when
handling dry DAB powder. Also, all glassware used during preparation of DAB solution should
be cleaned with bleach.
Reference: Rhodes et al., J Neurosci 15:5360, 1995 (https://www.ncbi.nlm.nih.gov/pubmed/7623158)

Transcardial Perfusion of Mice for Immunohistochemistry
Work in a fume hood using a perfusion stage and collection pan that will collect the perfusate (i.e.,
any formaldehyde-containing solutions) and allow for its proper disposal as per institutional
guidelines. This protocol is for use on mice weighing between 15 g and 25 g.
Anesthesia.
1) Weigh the mouse to calculate accurate anesthetic dosage and administer appropriately (e.g.,
100 mg/kg Sodium Pentobarbital in Fatal Plus Solution, Vortech Pharmaceuticals) via
intraperitoneal injection.
2) Check for complete anesthetic state (e.g., loss of corneal reflex by lack of blinking when air is
blown into eyes and loss of pedal pain reflex by lack of movement of paw/tail when squeezed).
3) The mouse should be completely anesthetized after ≈10 min. If the mouse still reacts to eye
blink/pedal reflex after 10-15 min, then administer additional anesthetic (e.g., 100 mg/kg Sodium
Pentobarbital in Fatal Plus Solution). Note the degree of reaction observed and repeat every 5
min until complete anesthesia is achieved.
Surgery/Perfusion.
4) Place the mouse on perfusion stage in collection pan and pin limbs to allow for exposure of
peritoneal cavity.
5) Use forceps in one hand to grab the skin over the xiphoid process and use scissors in the other
hand to cut, parallel to the spine, a patch of skin to reveal the outer abdominal wall.
6) The xiphoid process should now be visible. Use forceps in one hand to lift it, and use scissors
in the other hand to cut into and through the abdominal wall laterally, taking care to avoid cutting
any organs or major vessels.
7) The diaphragm should now be visible. Cut through diaphragm laterally, taking care to avoid
cutting any organs or major vessels.
8) Cut through the ribs and parallel to the lungs to create a chest "flap".
9) Retract chest "flap", fold it over the head and clamp it in place with a hemostat.
10) Use forceps in one hand to grasp the heart near its apex, use scissors in the other hand to
make an incision into the left ventricle and insert into the incision a 25 gauge needle attached to
a peristaltic pump via silicon tubing.
11) Clamp needle in place using a small hemostat.

12) Begin rapid perfusion of 1X PBS perfusate containing heparin (recipe below). Turn on peristaltic
pump at a rate of 7 mL/min (e.g., 25 rpm on a Watson Marlow 323 peristaltic pump with 1.6 mm
I.D. silicon tubing, I.V mini drip set) and begin perfusion of 11 mL (≈2 min) of ice-cold (4oC) 1X
PBS perfusate. Once perfusion has begun, cut the right atrium to allow drainage.
13) Switch perfusate to ice-cold (4oC) 4% formaldehyde (recipe below) and perfuse 25 mL at 5
mL/min (≈5 min). The upper body of the mouse should contract following the switch to fixative;
this is a sign of the correct needle position.
14) After perfusion of the mouse is complete, perform guillotine decapitation just behind the ears
and remove the skin and bone to expose the brain, taking care to minimize damage to tissue.
15) Remove the skull by chipping away with bone rongeurs.
16) Insert a spatula between the ventral side of brain and the bottom of skull to cut nerves and
scoop out the brain, taking care to clear away meninges and avoid slicing through the tissue,
especially near the olfactory bulbs.
Cryoprotection.
17) Place fixed brain in a 50 mL conical tube containing ice-cold (4oC) 10% sucrose solution (recipe
below) and incubate at 4°C overnight. The brain should equilibrate completely, as evidenced by
its sinking to the bottom of the tube.
18) Replace 10% sucrose with ice-cold (4oC) 30% sucrose and allow the brain to equilibrate
completely at 4°C, as evidenced by its sinking to the bottom of the tube. This may take as long
as three days.
19) Hemisect the brain by cutting through the midline with a fresh blade (razor or scalpel). Flash
freeze by placing hemisphere medial side down on a block of dry ice and covering with
pulverized dry ice.
20) Section immediately on a freezing stage microtome or wrap frozen hemispheres in plastic wrap,
then in aluminum foil and store at -80°C until sectioning.
Recipes
10X PBS Stock Solution (1 L)
2.3 g KH2PO4 17 mM (MW 136.09)

7.4 g Na2HPO4 52 mM (MW 141.96)
87.7 g NaCl 1.5 M (MW 58.44)
Allow reagents to dissolve into ≈900 mL of ddH20, adjust pH to 7.4 and bring to final volume of 1 L.

1X PBS Perfusate (1 L)
1) Dilute 100 mL of 10X PBS Stock Solution into 900 mL of ddH20.

2) Adjust pH to 7.4 with concentrated NaOH or HCl as needed.
3) Vacuum-filter solution through Whatman #2 paper over a Buchner funnel.
4) Add 10,000 units of heparin (e.g., ≈54 mg of 185.8 units/mg stock, Akron Biotech) and mix.
5) Chill to 4°C and keep on ice when in use.
0.4 M PB Stock Solution (2 L)
91.37 g Na2HPO4 320 mM (MW 141.96)

20.98 g NaH2PO4 ˑ H2O 76 mM (MW 137.99)
Allow reagents to dissolve into ≈1600 mL of ddH20, adjust pH to 7.4 and bring to final volume of 2 L.
For 0.2 M (fixative solution) or 0.1 M (sucrose solutions), dilute accordingly in ddH20 and check that
pH is 7.4.
Freshly Prepared 4% Formaldehyde (from Paraformaldehyde Powder) Fixative Solution (1 L)
1) Heat ≈400 mL of ddH20 to ≈60°C on a heated stirring plate in chemical fume hood (do not exceed
65°C as this will cause degradation of formaldehyde).
2) Add 40 g of paraformaldehyde.
3) Slowly add 10N NaOH dropwise and stir until solution becomes clear. Monitor pH, which should
not exceed pH 10, as this will cause degradation of formaldehyde.
3) Add 500 mL of 0.2 M PB.
4) Adjust pH to 7.4.
5) Vacuum-filter solution through Whatman #2 filter paper over Buchner funnel.
6) Adjust final volume to 1 L with ddH20.
7) Chill to 4°C and keep on ice when in use.
Sucrose solutions (100 mL)
1) For a 10% sucrose solution, dissolve 10 g of sucrose in ≈70 mL of 0.1 M PB. For a 30% sucrose
solution, dissolve 30 g in ≈70 mL of 0.1 M PB.
2) Check that pH is 7.4 and bring to final volume of 100 mL with 0.1 M PB.
3) Store at 4°C.
References: Rhodes et al., J Neurosci 15:5360, 1995 (https://www.ncbi.nlm.nih.gov/pubmed/7623158);

Manning et al., PLoS One 7:e38313, 2012 (https://www.ncbi.nlm.nih.gov/pubmed/22675541);
Bishop et al., J Neurosci 35:14922, 2015 (https://www.ncbi.nlm.nih.gov/pubmed/26538660).

Congo red (1% aqueous) Solution S067
Congo red (1% aqueous) Solution is recommended as negative stain for bacteria and spirochaetes.
Composition**
Ingredients
Congo Red 10 gm
Distilled water 1000ml
**Formula adjusted, standardized to suit performance parameters
Directions
1. Place a drop of Congo Red solution on a clean slide.
2. Mix one or two loops of the microorganism/ culture into the drop of Congo red solution.
3. Allow the smear to air-dry. NOTE: Do not heat fix the smear.
4. Rinse the smear gently with tap water and blot dry.
5. Examine the smear under oil immersion objective.
Principle And Interpretation
Negative Staining Techniques work in a manner opposite to simple techniquer. Bacteria are mixed on a slide with an acidic
dye such as congo red or the black stain, nigrosin. Bnmf n qdc hr sgd chrncht l r` ks ne chogdmxk,chr` yn,ahr,0,m` ogsgxk` l hmd,3,rt kenmhb
` bhc' 0(-The mixture is smeared across the face of the slide and allowed to air dry.
Because the stain carries a negative charge, it is repelled by the bacteria, which also have a negative charge. The stain gathers
around the cell which often appears larger than stained cells.
Quality Control
Appearance
Intense red solution.
Clarity
Clear without any particles.
Microscopic Examination
Negative staining is carried out. Staining characteristics of organism is observed under microscope by using oil immersion
lens.
Results
L hbqn nqf ` mhrl r , Colourless
A` bj f qnt mc , Pink
Storage and Shelf Life

Store below 30°C in tightly closed container and away from bright light. Use before expiry date on label.
Reference 0( Ahnknf hb` k Rs` hmr,9th edition, R.D.Lillie, M.D., E.H.Stotz,Ph.D.,V.M. Emmel ,Ph.D., M.D.
Revision : 1 / 2016
Disclaimer :
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work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
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Perfusión Normal
Conviene que el día antes de la perfusión se compruebe llenando el sistema de perfusión

con agua que no hay fugas y que todo marcha bien, así de paso se limpian los reciientes
y tubos.
MATERIAL
1. Sistema de perfusión.
2. Caja con reja para sujetar el animal.
3. Fijador. 4%PF en PB 0,1M (pH 7,2 -7,4). Es mejor prepararlo fesco.
Volumen a usar: peso del animal en gr .(Cuando no se perfunde la parte inferior del
animal, de lo contrario seria peso x 1,5)
Volumen a preparar: volumen de uso y 100 ml más por rata para postfijación
Ejem para 300 ml:
Calentar hasta 70ºC un volumen de 200ml de dH2O en microondas y colocar en
un agitador caliente en la campana apagada, añadir 12gr de PF y agitar, encender
la campana, añadir dos o tres gotas de NaOH 1N y tapar, agitar hasta que este
cristalino. Filtrar en una probeta, y cuando esté algo más frío añadir 30 ml de PB
y 24ml de Glutaraldehido (Viene en stock al 25%, queda a concentración de 2%
en los 300ml) y enrasar con dH2O hasta un volumen final de 300ml. Medir pH.
4. Salino. 0,9% ClNa en dH2O.(0,9 gr en 100ml de dH2O)
5. Instrumental quirúrgico. Tijeras, pinzas, bisturí, porta bisturí, tijeras clampar,
gubia
6. Instrumental de perfusión. Cánulas, agujas (grises), jeringuillas de insulina.
7. Botes de orina 100 ml.
8. PB 0,1 M
9. Hielo
10. Hojas seguimiento animales. Numeración , A(Ana)01(año)01(nº animal)
11. Anestesia. Hay varias posibilidades de anestesia y además no son las
mismas dosis para rata que para ratón. Leer el documento de anestesia en el
que se explica todo. Lo mas habitual es utilizar una anestesia de Ketamina
(Ketolar)y Metedomidina (Dormtor). De Ketolar: 75mg/Kg peso para ratón o
50mg/Kg peso para rata y de Dormtor 1mg/Kg peso del animal.
Dormtor). El Ketolar y el Dormotor se mantienen tapados y en nevera.
Para rata sería 0,1 ml Ketolar (stock 50mg/ml) por cada 100 gr de peso animal y para
ratón 0,15 ml Ketolar por cada 100 gr de peso mas un poquito de Dormtor.
PREPARACION DEL SISTEMA DE PERFUSION
1. Colocar el sistema de perfusión con el pie encima de la mesa, llenar toda la vía
del fijador y eliminar las burbujas, cambiar la “T” y pasar el salino y eliminar
las burbujas.
PERFUSION
1. Anestesiar al animal. Intraperitoneal. Coger al animal del pescuezo y las patas

delanteras sin apretar salvo que se mueva. Colocarlo con el vientre hacia arriba,
retirando el rabo hacia atrás, y pinchar la dosis de anestesia. El lugar ideal seria,
en la mitad de una de las partes que separa la línea alba y más arriba de la vejiga
formando un ángulo de 45º.
2. Colocar el animal en la reja y cortar la piel con un bisturí siguiendo la línea alba
y retirarla dejando al descubierto desde la traquea hasta el abdomen bajo.
3. Con pinzas y tijeras, cortar desde el abdomen hacia los lados y hacia la parte
superior del animal, dejando al descubierto los intestinos que se retiran a un lado
para clampar lar arterias que van a las extremidades inferiores, de esta forma
solo fijamos la parte superior que es la que nos interesa y así usamos menos
fijador. Atención, las tijeras se introducen bastante para cortar mas superficie en
menos tiempo.
4. Cuando ha quedado al descubierto el esternón, se levanta con las pinzas de
modo que podemos cortar con cuidado el diafragma y así dejar al descubierto el
corazón, desde este momento todo ha de ser muy rápido ya que entra en hipoxia.
5. Cortamos lo mas lateral posible las costillas, y se coloca una tijera de clampar en
el esternón para mantener las costillas retiradas del corazón.
6. Con unas tijeras pequeñas y de punta se corta la grasa circundante del corazón y
se retira el timo para poder ver la aorta que tenemos que canular.
7. Sujetamos el corazón con unas pinzas y hacemos un corte en el ventrículo
izquierdo. Para ratas pequeñas no es necesario cortar.
8. Meter la cánula con goteo suave de salino en dirección a la aurícula derecha pero
sin llegar a la aorta, regulamos el flujo de salino.
9. Canulamos la aorta
10. Cortamos la aurícula derecha. Dejamos pasar el salino un tiempo suficiente.
Máximo 2 min.
11. Cambiar la ”T” para meter el fijador. En este momento el movimiento de patas
delanteras y cabeza es señal de que el fijador esta entrando bien, ir comprobando
la dureza de las patas y el ritmo de goteo ya que éste disminuye con el tiempo.
Finalizado el proceso desconectar.
NOTA: la perfusión de la médula puede ser por inmersión, podemos seguir clampando
abajo. A veces se perfunde sin lavar con salino.
POSTFIJACION A 4ºC
1. Cortar la cabeza, se puede cortar con una guillotina, si no, primero con bisturí
luego con las tijeras, a la altura del comienzo de las patas delanteras.
2. Pelar la cabeza y abrir entradas en el cráneo a la altura de los ojos y el inicio de
los bulbos con la gubia o pinzas si es un animal joven. Extraer el cerebro y
mantener toda la noche a 4ºC en un falcon con fijador hasta arriba.
3. Al día siguiente
a. Criostato: crioproteger los cerebros en fijador con 30% sacarosa hasta
que se hundan. Congelar los cerebros en hielo seco.
b. Vibratomo: lavados con PB 0,1M a 4ºC una vez al día durante cuatro
días. Luego pasar a PB 0,1 M con azida al 0.05%.
NOTA: un buen indicativo de una buena perfusión es que la hipófisis esté blanca, ya
que es una glándula muy irrigada.
Fecha de creación 23/10/02 Autor: Marina, revisado Agosto 2012
Protocolo Inmunohistoquímica Laboratorio Comunicación Intercelular
Para tejidos por congelamiento:
Fijar los tejidos con paraformaldehído al 4%, posteriormente los tejidos son tratados
por un día con sacarosa al 10%, 20%, 30%. Y finalmente estos tejidos son montados
en el medio de montaje O.C.T para ser cortados en el criostato. Los tejidos cortados
fueron dejados en una placa multipocillo con una solución de PBS-1X.
Se realizaron los siguientes tratamientos:
1. Eliminación de la autofluorescencia: Los tejidos son tratados con un solución

de PBS-1X y 3% de NaBH4 durante 24 hrs. Se realizan dos lavados de 10
minutos con PBS-1X.
2. Recuperación antigénica: Los tejidos son montados en portaobjetos que

contienen poli-lisina (Nota: Los portaobjetos son preparados un día antes de su
utilización) estos se dejan secar durante 24 hrs a temperatura ambiente.
 Transcurrido este tiempo, se utiliza una solución de Citratro 0,01 M a

pH: 6.0 la cual es calentada en un vaporera hasta llegar a los 95ºC. Al
llegar a la temperatura óptima las muestras son tratadas con agua
destilada por 15 minutos y luego con citrato durante 5 minutos todo esto
a temperatura ambiente.
 Terminado este proceso, las muestras son introducidas a la solución de

citrato que se encuentra a 95ºC, se deja por 30 minutos. Luego las
muestras son enfriadas en agitación por 20 minutos. Se realizan dos
lavados de 10 minutos con agua destilada en agitación.
3. Eliminación Peroxidasas: Las muestran son tratadas con una solución para
eliminar la peroxidasas (200 mL de Metanol con 13,3 mL de peróxido de
hidrógeno para que quede al 30%) durante 30 minutos. Finalizado este proceso
se realizan dos lavados de 10 minutos con agua destilada en agitación.
Realizados estos tratamientos se procede a bloquear y permeabilizar los tejidos con

las siguientes soluciones:
1. Para anticuerpos ABCAM: “Panx1(Rb), Panx2(Rb), Gephyrin(Ms), P2X7(Rb)”

Contiene 1% de BSA, 10% de GS, 0,3 M de Glicina en PBS-Tween al 0,1% en
PBS-1X, durante 1 hora y 30 minutos, o por 24 horas.
2. Para anticuerpos NeuroMab: “GluA1(Ms), GluA2(Ms), GluN2B(Ms), NR1(Ms),

NR2A(Ms), mGlu1/5(Ms) Sap102(Ms)”.
Contiene 10% GS, 0,3 % Triton X-100 en PBS-1X, 1 hora por agitación a 4ºC.
3. Para anticuerpos Millipore: “PSD-95 (Ms)”

Contiene 5% SFB y 0,1% Triton X-100 en PBS-1X.
4. Para anticuerpos Promega: “β-Tubulina III (Ms)”
Contiene 1% GS, 0,1 % Triton x-100 en PBS-1X, durante 1 hora a 4ºC.
5. Para anticuerpos Santa Cruz: “Panx1(Gt), Syp (Gt)”

Contiene 3%BSA, 0,1% Triton X-100 en PBS-1X durante 1 hora a 4ºC.
6. Para anticuerpos Invitrogen: “Panx1(Rb), Cx26(Ms)”

Contiene 6% GS, 3% HS en Tritón 0,1% en PBS-1X durante 1 hora a 4ºC.
7. Para anticuerpo inmunoprecipitado: “Cx26(Rb)”

Contiene 3%BSA, 6%GS en Tritón 0,1% en PBS-1X durante 1 hora a 4ºC.
Cada una de las soluciones de bloqueo-permeabilización es agregada a los

portaobjetos durante dos horas en agitación, o también toda la noche a 4ºC.
Los anticuerpos primarios son preparados dependiendo de la solución de bloqueo de

cada anticuerpo, son dejados incubando durante 24 horas en una cámara húmeda a
4ºC.
Terminada la incubación con los anticuerpos primarios se procede a lavar los

portaobjetos con las soluciones correspondientes a cada anticuerpo, lavados de 10
minutos durante 1 hora.
1. PBS-1X con 0,1% de Tritón X-100.

2. PBS-1X con Tween-20 al 0,1%, Glicina 0,3 M.
Luego de los lavados los tejidos son incubados en una cámara húmeda con los
anticuerpos correspondientes preparados en las soluciones de bloquea específicas
para cada anticuerpo. Se dejaron durante dos horas en la cámara húmeda a 4ºC.
Finalmente se realizaron lavados de 10 minutos durante una hora, y luego se agrega

el medio de montaje (Dako) y se colocan los cubreobjetos.
Diluciones Anticuerpos
1. Anti-β-tubulina III Ms: 1:500

2. Anti-Panx1 Rb: 1.500
3. Anti-PSD95 Ms: 1:300
4. Anti-GluN2A Ms: 1:200
5. Anti-GluN2B Ms: 1:200
6. Anti-P2X7 Rb: 1:300
Protocolo procesamiento del tejido para histología (AG Enero 2012)
1. Perfundir la rata anestesiada a través del ventrículo izquierdo con 4% paraformaldehido en 0.1M PB
(pH 7.2-7.4) preparado en el momento.
Si no se perfunde y se extrae directamente el cerebro empezar aquí:

2. Extraer el cerebro y postfijar 12h/over night a 4ºC en 4% PF en 0.1M PB.
3. Crioproteger el tejido durante 24-48h (hasta que se hundan totalmente, no suelen necesitar mas de
24h si son hemicerebros) en una solución de 30% sacarosa-4% PF en 0.1M PB.
NOTA: Dejar el tejido demasiado tiempo en fijador no es bueno porque el tejido se encoje y los
epítopos para inmunohistoquímica pueden perderse.
4. Montar en un papel de filtro con OCT y congelar rápidamente en nieve carbónica en polvo. Envolver
los cerebros en reynolon y guardar a –70ºC.
En un trocito de papel de filtro se marca con lápiz el nombre del animal y por el lado contrario se
pone una gota de OCT sobre la que se pone el cerebro ya orientado para su posterior corte al
criostato, este montaje se coloca sobre hielo seco machacado y se cubre el cerebro totalmente con
hielo seco en polvo, a los 10 min ya se puede envolver en reynolon y meter rápidamente a -70 hasta
que se seccione con el criostato.
5. Cortar en el criostato a 25m de grosor. Usar inmediatamente o recoger en CBS y congelar a -70ºC.
PB 1M pH 7.2-7.4
610ml Na2HPO4 1M autoclavado (disolver com agua caliente)
390ml NaH2PO4 1M autoclavado
ajustar pH con NaOH 10N en el PHímetro
4% PF (por litro)
40g paraformaldehido
Disolver en 700ml dH2O precalentada a 65ºC (es muy importante no pasar de esta temperatura pues si no el
Paraformaldehido cambia y no fija igual). Añadir unas gotas de NaOH 1N. Añadir 100ml de PB 1M y enrasar a
1000ml. Medir el pH (7.2-7.4, comprobar con una barita de PH) y filtrar con papel de filtro. Enfriar.
30% Sacarosa en PF
Disolver 30 gr de sacarosa/100 ml de Paraformaldehido al 4% en PB 0.1M (En un vaso de precipitados con imán).
NOTA: No se si se podría hacer todo con PBS de cocina de medios, como tu eres química te adjunto la receta del
PBS 10x y como verás la molaridad es 1/10 menor, lo acabo de ver
CBS (para 500ml)

30% glicerol (150ml)
30 % etilenglicol (150ml)
40% 0.1M tampón fosfato pH 7.2 (20ml de PB 1M + 180ml H2O)Mezclar y filtrar

Uc Davis/Nih Neuromab Facility: Immunofluorescence Staining of Free Floating Brain Sections

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UC Davis/NIH NeuroMab Facility

Department of Neurobiology, Physiology and Behavior, UC Davis, Davis CA 95616-8519

Immunofluorescence Staining of Free Floating Brain Sections

6) Wash sections with gentle rocking/agitation at 4ºC, 5 min in vehicle.

7) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.1 M PB.

8) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.05 M PB.

9) Mount on gelatin-coated microscope slides (floating in 0.05 M PB in a 15 ml Petri dish).

10) Air dry

12) Seal with nail polish.

Immunofluorescence Labeling of Free-Floating Perfusion-Fixed Brain Sections

8) Sudan Black B treatment (optional, see below).

last updated 14Oct2016 page 1 of 2

1) Completely rehydrate slide-mounted sections with ddH20

2) Completely submerge slide in a tray containing 0.05% SBB (recipe below).

5) Air dry and proceed to coverslipping step.

Refs: Oliveira et al., Histol Histopathol 25:1017, 2010 (https://www.ncbi.nlm.nih.gov/pubmed/20552552);

last updated 14Oct2016 page 2 of 2

Antigen Retrieval Methods for Free Floating Brain Sections

4) Wash sections 2X in cold 0.1M PB

Immunoperoxidase Labeling of Free-Floating Perfusion-Fixed Brain Sections

last updated 14Oct2016 page 1 of 2

DAB/NAS solution (100 mL, prepare immediately before use):

Reference: Rhodes et al., J Neurosci 15:5360, 1995 (https://www.ncbi.nlm.nih.gov/pubmed/7623158)

last updated 14Oct2016 page 2 of 2

Transcardial Perfusion of Mice for Immunohistochemistry

11) Clamp needle in place using a small hemostat.

last updated 14Oct2016 page 1 of 3

15) Remove the skull by chipping away with bone rongeurs.

10X PBS Stock Solution (1 L)

2.3 g KH2PO4 17 mM (MW 136.09)

last updated 14Oct2016 page 2 of 3

1) Dilute 100 mL of 10X PBS Stock Solution into 900 mL of ddH20.

0.4 M PB Stock Solution (2 L)

91.37 g Na2HPO4 320 mM (MW 141.96)

Freshly Prepared 4% Formaldehyde (from Paraformaldehyde Powder) Fixative Solution (1 L)

Sucrose solutions (100 mL)

References: Rhodes et al., J Neurosci 15:5360, 1995 (https://www.ncbi.nlm.nih.gov/pubmed/7623158);

last updated 14Oct2016 page 3 of 3

Storage and Shelf Life

Conviene que el día antes de la perfusión se compruebe llenando el sistema de perfusión

PREPARACION DEL SISTEMA DE PERFUSION

1. Anestesiar al animal. Intraperitoneal. Coger al animal del pescuezo y las patas

Para tejidos por congelamiento:

Se realizaron los siguientes tratamientos:

1. Eliminación de la autofluorescencia: Los tejidos son tratados con un solución

2. Recuperación antigénica: Los tejidos son montados en portaobjetos que

 Transcurrido este tiempo, se utiliza una solución de Citratro 0,01 M a

 Terminado este proceso, las muestras son introducidas a la solución de

Realizados estos tratamientos se procede a bloquear y permeabilizar los tejidos con

1. Para anticuerpos ABCAM: “Panx1(Rb), Panx2(Rb), Gephyrin(Ms), P2X7(Rb)”

2. Para anticuerpos NeuroMab: “GluA1(Ms), GluA2(Ms), GluN2B(Ms), NR1(Ms),

3. Para anticuerpos Millipore: “PSD-95 (Ms)”

5. Para anticuerpos Santa Cruz: “Panx1(Gt), Syp (Gt)”

6. Para anticuerpos Invitrogen: “Panx1(Rb), Cx26(Ms)”

7. Para anticuerpo inmunoprecipitado: “Cx26(Rb)”

Cada una de las soluciones de bloqueo-permeabilización es agregada a los

Los anticuerpos primarios son preparados dependiendo de la solución de bloqueo de

Terminada la incubación con los anticuerpos primarios se procede a lavar los

1. PBS-1X con 0,1% de Tritón X-100.

Finalmente se realizaron lavados de 10 minutos durante una hora, y luego se agrega

1. Anti-β-tubulina III Ms: 1:500

Si no se perfunde y se extrae directamente el cerebro empezar aquí:

Disolver 30 gr de sacarosa/100 ml de Paraformaldehido al 4% en PB 0.1M (En un vaso de precipitados con imán).