I'm doing IHC with frozen sections. I blocked with 5% horse serum in PBS but got very strong fluorescent background (both green and red). I found a lot of blocking reagents available from companies. (but I still have to add the serum myself). Is it necessary to purchase the reagent from the companies? Any recommendations? What do they actually use? Thanks!
In addition to Haobo Li's good suggestion above, be sure to add 0.05% Tween 20 (a surfactant) to your blocking buffer. If your secondary Ab is made in donkey, use 3 to 5% donkey serum in your blocking buffer, if your secondary is made in rabbit, use 3 to 5% normal rabbit serum etc. Sometimes 3% BSA in your blocking buffer works the best for no known reason. Also a combination of 5% normal goat serum and 5% normal swine serum helps clean things up very well. It is often an empirical endeavor - until it works. Lately, I use TRIS Buffered Saline (TBS pH 7.4) - I buy 10X TBS from Bio-Rad, then, make a 1X solution containing 3% BSA and 0.05% Tween 20. It depends on what species your primary and secondary Abs are made in to the most extent, but then it is trial and error after that. Most blocking buffers are a PBS or TBS buffer of physiologic pH, with addition of 0.05-0.1% of a surfactant (e.g., Tween 20, NP-40, Triton X-100) and 3 to 5% of a protein source of some kind (casein, some kind of normal animal serum, BSA, sometimes gelatin, acetylated BSA and so on). Much cheaper to make your own.
Thank you guys. Yes I've heard of the serum of host where the sec antibody is from. But I have two primary and two secondary antibodies, so I need two serum maybe...I just tried 20% FBS and at least it's much better than the 5% horse serum I used before. My sec antibodies are from gt and dk. Is TBST better than PBS? Keep trying.
my answer is similar to Haobo. 10% normal serum of secondary antibody species + 0.3% triton-X 100 + 0.1M PBS (I use PBS tablets from Sigma Aldrich). GOOD LUCK
I use TBST or TBS + Triton-X 100 (0.3-0.5%). For serum (10%) I would try either of the 2 your 2aries were raised in and a combination as well. I wouldn't bother with the company-made blocking reagents.
I am still not sure why PBS vs TBS would matter. But the TWEEN/Triton-x is there to penetrate the tissue to allow access of the blocking solution. I would recommend 10-20% serum in the block. Also I recommend filtering the wash buffer, block, and antibody diluent before use. .22 micron filter.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts