Hello fellow researchers
I am trying to stain cryostat-sectioned spleen tissue, using free-floating chromogenic immunohistochemistry. Unfortunately, I'm not able to detect any staining (MHC class I) on my mounted slides. My approach is as follows:
1. Fixation of the spleen in 10% natural buffered formalin for 24 hrs
2. Dehydrate in 30% sucrose (w/v) in 0.1M Phosphate Buffer for 24 hrs
3. Sectioning the spleen in Cryostat-microtome, sections 45-80 um (pretty thick, due to fragility)
4. Put in preheated sodium Citrate - pH 6.0 (Retrieval buffer), in 80 degree celcius for 30 minutes.
5. Block endogenous peroxidase in 1% H2O2 in PBS
6. Block mouse Fc-receptors (1% mouse serum) in TX-PBS
7. Block with secondary antibody host serum (5%) in BSA-TX-PBS
8. Primary ab incubation on shaker (4 degree celcius overnight) in BSA-TX-PBS
9. Secondary ab-biotin incubation on shaker (RT in 60 min) in BSA-TX-PBS
10. Avidin:Biotin:Peroxidase Complex solution incubation (RT in 30 min) in TX-PBS
11. Chromogenic solution untill thoroughly stained.
12. Mounting on slides, let dry (but not too dry, due to curling) - add DPX.
If you have any surggestions or ideas for troubleshooting, it will be much appreciated :).
Hi Nicolai
You can try two modification due to high section thickness first, you can try to permeabilize tissue after antigen retrieval step by incubating section in 2-3% triton x 100 in phosphate buffer saline for 30 mins (you can do this with peroxidase blocking step just by adding H2O2 in this solution and also i generally prefers to add H2O2 at 3% concentration) this will help antibody to penetrate in the tissue section second, thing you can try is to incubate sections with primary antibody for longer duration (close to 3-4 days).
Vikas
Add triton X into your antigen retrieval & Switch to EDTA ph 8, this will open up more epitopes but will also open up more endogenous biotin. So you will need to add a endogenous biotin blocker before using the ABC complex. Also incubate overnight at RT on a shaker if you can. Also add washes with TX in between steps. Do all steps on shaker if you can.
Thinner sections (30µm) are highly recommanded. 3% H202 in PBS for 20 min is okay. Instead of antigen retrievel use dilution medium which contains 0,2%Triton-X100 in PBS or TBS.
For the blocking step add 10% normal serum (depnds on the host animal of the secondary antibody) and 20% avidin or streptavidin (if you are working with ABC or strepavidin labeled with POX). the Avidin-Biotin-Blocking Kit from Vector Labs makes a good job. For the primary antibody , secondary ab and detection system use 1% normal serum in 0.2 % Triton X-100. Use different dilutions of the primary antibody to find out which one is the best for your protocol. Reaction time and temperature: Blocking , secondary and detectionsystem 90 min each at room temperature. The primary antibody over night at room temperature as well. Good luck!
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