F4/80 staining in mouse liver

[Philip]

Hi,
can anyone help me with the staining for F4/80  (Mouse macrophages, Kupffer cells) in the liver. I tried several different antigen retrieval techniques  with no success. I'm using primary antibody from serotec and using the vectastatin ABC rat kit. the staining works for frozen sections But i'm not getting any stain for the formalin fixed paraffin sections. I usually fix the sections in 10% NBF for 24 h. please help.

[excalibur]

I have done F4/80 on mouse FFPE tissue using citrate antigen retrieval in a rice steamer.

[Philip]

I tried citrate buffer pH6 on a hot plate at 100 degree celsius for various time points ranging from 10 min to 40 min. did not get any stain at all. I think something had gone wrong during the embedding stage. did u get good staining with citrate buffer. can please give me suggestions about the embedding temperature. I've a gut feeling that the high temperature during paraffin embedding is killing the antigen.

[excalibur]

If your paraffin temp at any stage was above 60C you probably killed the antigen.

[raintxy]

Hi, I am also using this F4/80 (serotec) for IHC on paraffin section of mice intestine and whole mount of mice intestine muscularis layer.
For whole mount, i use 2nd Ab AlexaFluor 488, initially i use 1:100 dilution of F4/80, and cannot see any positive staining of the resident macrophage. then i checked other's papers and use higher concentration 1:20 and 1:50, and scan the whole mount (25uM thickness) about 1uM 1 slide, then i found that it only stained the outside layer. i also used this antibody for IHC on paraffin section at 1:50, using Vector's ABC kit, the background is very high, so difficult to tell which cell is positive.
to solve the problem, i borrowed another F4/80 which is also from Serotec from another lab, it was bought 10 years ago, at 1:50 dilution the old one works although the background is a little bit higher, but at least it can stain the macrophages inside. the 2 antibodies gave me totally different image

[Philip]

Hi Paula

I think you are right. I might have killed the antigen during the paraffin embedding. I'm going to try embedding with low melting point paraffin. Do you suggest any particular low melting paraffin from any vendor. also how much time you kept the slides in citrate buffer and at what temperature for the antigen retrieval.
thanks

[excalibur]

Your paraffin may be fine. Most paraffin sold for histology have melting points in the range needed. What are you using now? You may just need to make sure your equipment is not over heating it and is set for 58-60C.

I place my slides in a plastic staining container with the citrate buffer at room temp.
I then place the container and all in the rice steamer and set it for 60 mins. After the 60 mins, I let it sit with the lid off for 25 mins. Then add tap water slowly to further cool.

If you go to IHC world's home page, many protocols are listed, including this and other antigen retrieval methods.