I have stained liver and kidney tissue several times by following method but I could not get any results. Does anyone have any suggestion?
The protocol that I use is:
1) heat-mediated retrieval in Tris-EDTA (pH 9.0) buffer.
2) Remove from heat and let stand at room temperature in buffer for 20 minutes.
3) Rinse tow times in TBST 5 minute each.
4) Add Triton-X100 for10 minutes
5) Wash slides in 1X TBST 2 times for 5 minutes
6) Add blocking solution (Avidin and Biotin Solution / 10 minutes each)
7) Wash slides in 1X TBST 2 times for 5 minutes
8) Add Hydrogen Peroxide (3%). Incubate 10 minutes at room temperature.
9) Wash slides in 1X TBST 2 times for 5 minutes
10) Add blocking solution (10% Normal Goat Serum in TBST)
11) Apply 100-200 µl per slide of diluted primary antibody at recommended concentration (used different concentration 1:100, 1:500, 1:1000 and 1:2000). Incubate overnight at +4 C (Dilution: in 1% NGS in TBST).
12) Wash slides in 1X TBST 3 times for 5 minutes
13) Apply a 1:200 µl per slide of diluted secondary antibody. Incubate for 60-30 minutes at room temperature (Incubation time checked for both times).
14) Wash slides in 1X TBST 3 times for 5 minutes.
15) Apply a 100-200 µl per slide of ABC System (recommended concentration: 1:100, 1 ul A solution, 1 ul B solution).
16) Wash slides in 1X TBST 3 times for 5 minutes
17) Add 100-200 ul of DAB as chromogen (15-60 second)
(Do not allow tissues to dry at any time during the staining procedure).
I'd recommend a red chromogen (AEC instead of DAB)- simply because it will be easier to see and quantify the macrophages. An alternative marker may be CD11b.
I do this stain on a regular basis. Here is the stream-lined version that I use:
1) low pH heat-retrieval in a citrate buffer
2) H2O2 blocking for 5 min, avidin/biotin and protein blocks, 10 min ea
3) F4/80 1:100 for 30 min
4) D-a-Rat 1:100 15 min
5) LSAB2-SA, 15 min
6) DAB development, 10 min (hemotoxylin counter stain)
TBS wash between each step of course ( I just use two fresh baths of TBS, fully flush slides, remove and continue).
One thing is to be sure you have a good positive control!
We do not use triton-X in our protocol. I have found older sections do not stain as well as freshly cut ones.
I also notice the same issue that older sections are difficult to stain. However, in my case, I parallelly stained the same batch of sections with F4/80 and vimentin. Vimentine was stained well, but F4/80, I did not get any staining at all. So I am wondering if F4/80 staining requires any special processing.
I have tried F4/80 by immunoflource staining and I got good results but never get result by IHC
You might try increasing the antigen retrieval time from 20 min to 40 min. This works for older tissues that I often get in TMAs
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