Two things can help with PFA. 1) stabilize the antigen using glycine (I think 3%) after antigen retrieval. 2) Do not use methanol (if you are) while treating with H2O2 for quenching endogenous peroxidase. Hope this helps.

Thank you Anuj, I will try the glycine. Gor how long do you recommend me to incubate it?

It should be 0.6% Glycine in PBS for 10 min. This step will be right after the antigen retrieval step.

Hi Andrea!

This i my "favorite-protocol"….

Freeze tissue in -55°C isopentane (cool it down using liquid nitrogen or dry ice). Section 6µm thin. Fix tissue  slide for 10 min. in 4% PF. Rinse in PBS twice for 2 min. Add 5% goat or mouse sera (Jackson Immunoresearch) diluted in PBS 20 min. Tap of excess sera and add primary antibody (I would recommend BDs antibodies instead), incubate for 60 min. A dilution of 5µg/ml in PBS usually give a nice staining, without background. Rinse in PBS, twice for 2 min. Add sec. antibody - for example Jackson Immunoresearch secondary antibodies - biotinylated goat anti…. - what ever your primary is made in. Incubate for 30 min. Rinse as above. Add streptavidin-peroxidase, also from Jackson, incubate for 30 min. Rinse as above. Develop with AEC (DAKO), watch the development under the microscope - usually 5 min. Rinse in tap water, counterstain with HTX. Mount in water-based mounting media (DAKO). 

Or - if you prefer fluorescence - add a secondary Alexa 488 goat-anti….5µg/ml for 30 min, rinse in PBS, counterstain with Pro-Long gold anti-fade with DAPI (Molecular Probes).

Good Luck!!

For Frozen Section: 1 After total drying section, Fixation in 1:1 Methanol:Aceton at -4C; 2  Donot antigen retieval .

Immunohistochemistry Protocol (Paraffin)

A. Solutions and Reagents

1.     Xylene

2.     Ethanol, anhydrous denatured, histological grade (100% and 95%)

3.     Deionized water (dH2O)

4.     Hematoxylin (optional)

5.     Wash Buffer:

1X TBS/0.1% Tween-20 (1X TBST): To prepare 1 L add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween-20 and mix.

10X Tris Buffered Saline (TBS): To prepare 1 L add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl.

6.     Antibody Diluent: SignalStain® Antibody Diluent #8112

7.     Antigen Unmasking: TE: 10 mM Tris/1 mM EDTA pH 9.0: To prepare 1L add 1.21 g Trizma® base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 950 ml dH2O. Adjust pH to 9.0 then adjust final volume to 1000 ml with dH2O.

8.     3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.

9.     Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.

10.   Detection Reagent: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (#8114)

11.   DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1.     Deparaffinize/hydrate sections:

1.     Incubate sections in three washes of xylene for 5 minutes each.

2.     Incubate sections in two washes of 100% ethanol for 10 minutes each.

3.     Incubate sections in two washes of 95% ethanol for 10 minutes each.

2.     Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

NOTE: This procedure describes the conditions that are recommended for the Biocare Medical Decloaking Chamber. Device-specific settings and operating instructions should be utilized for other pressure cookers.

1.     Place slides in 250 ml room temperature TE unmasking solution in a 24-slide holder.

2.     Place 500 ml dH2O into the pressure cooker.

3.     Place the slide holder into the pressure cooker, touching the heat shield. It may be advantageous to place a second 24-slide holder into the pressure cooker, filled with 250 ml water and blank slides.

4.     Seal the chamber and proceed with retrieval. Settings for the Biocare Medical Decloaking Chamber follow.

1.     SP1 125°C 30 seconds

2.     SP2 90°C 10 seconds

5.     Carefully vent the device, then remove the lid and cool the slides on the bench for 10 minutes.

6.     Rinse the slides with dH2O.

D. Staining

1.     Wash sections in dH2O three times for 5 minutes each.

2.     Incubate sections in 3% hydrogen peroxide for 10 minutes.

3.     Wash sections in dH2O twice for 5 minutes each.

4.     Wash section in wash buffer for 5 minutes.

5.     Block each section with 100-400 µl blocking solution for 30 minutes at room temperature.

6.     Remove blocking solution and add 100-300 µl primary antibody diluted in SignalStain® Antibody Diluent #8112 to each section. Incubate 1 hour at room temperature. Refer to product datasheet to determine the recommended dilution.

7.     Equilibrate SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 reagent to room temperature.

8.     Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.

9.     Add 1-2 drops of SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 to each section. Incubate 30 minutes at room temperature.

10.   Remove SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 solution and wash sections three times with wash buffer for 5 minutes each.

11.   Add 100-400 µl DAB or suitable substrate to each section and monitor staining closely.

12.   Upon completion of development, immerse slides in dH2O.

13.   If desired, counterstain sections in hematoxylin per manufacturer’s instructions.

14.   Wash sections in dH2O two times for 5 minutes each.

15.   Dehydrate sections:

1.     Incubate sections in 95% ethanol two times for 10 seconds each.

2.     Repeat in 100% ethanol, incubating sections two times for 10 seconds each.

3.     Repeat in xylene, incubating sections two times for 10 seconds each.

16.   Mount coverslips.

Thank you so much collegues, I will try all this ideas. 

This may be an odd question, but are you sure it is the protocol that's wrong and not the antibodies? For instance, is the problem that there's no staining, too much background staining, etc.? Have you tried different antibodies?

Dear Roxanne,  I'm in the process to find new antibodies too. Do you have any suggestion? Thank you. 

Here are examples of what we have used in our laboratory. Note that those were used with paraffin embedded tissues and a Ventana machine.

CD3:

rabbit anti-human CD3e, #A0452, Dako

rabbit anti-human CD3, #ab5690, Abcam

CD4:

mouse anti-human CD4, #IR649/IS649, Dako

mouse anti-human CD4, #ab846, Abcam

rat anti-human CD4, #sc-13573, Santa Cruz

CD8:

mouse anti-human CD8, #IR623/IS623, Dako

rabbit anti-human CD8, #ab4055, Abcam

mouse anti-human CD8, #sc-7970, Santa Cruz

Thank you so much. I'm working with mouse tissue infected with tuberculosis and I rather prefer to use antibodies that works in paraffin embedded tissues.  I appreciate your help. 

Dear Colleagues, I have also problem in Immunostaining of Parrafin sections of Rat sciatic nerve and spleen with CD3 and CD5. I am also using rabbit anti-human CD3e, #A0452, Dako as primary antibody and using Heat method for antigen retrival, but still i am unable to establish T cell staining. Please can any body gives some suggestion to improvise my stainings. Thanks