I am doing double immunohistochemistry staining of rat brain sections. Unlike most protocols, I work with thicker sections (40μm, it still works very well) with all incubations done in a tissue culture plate. I follow the same protocol every time, I mix the sections during all incubation steps and still I get different staining intensities every time! Sometimes even the two hemispheres of a single section are coloured differently! I tried to optimize the conditions in multiple ways - I tried 12-well, instead of 24-well plate, with higher volumes, more mixing, extended incubation times, less sections per well, etc., yet this doesn't improve enough, as I expected. I still can't achieve regular staining and more importantly - easily comparable between experiments! Even the final step - the substrate reaction - develops differently every time! Also, the background level is hard to assess. I guess I'm missing something, I will be glad if someone with more experience can give me a clue, or has encountered similar issues, to share some suggestions on how to improve.
I am staining for c-Fos and calbindin, biotinylated secondary antibodies + streptavidin-HRP. I use two different substrates - standard DAB and a two-component Vector SG peroxidase substrate kit. All concentrations should even be in surplus. Tissue preparation has been performed similarly from the start.
Thank you!
Hi Ivaylo,
for the free floating reactions I recommand to use the 24 well plates. Use 1 section/well and 500µl solution/well. If you like to react several sections simultaniously take 6 well plates and 2 ml solutions/well. You can easily react more then 3 sections/well. Put he plates on an rocking-table so that the sections continiously moved and rinsed. some importenant blocking steps are necessary when you are working with Strpavidin and DAB, because this steps are very often responsible for unspecific backgroundstaining. 1. you have to block the endogenious peroxidase with H2O2 (30% H2O2 1:100 in PBS). 2. unspecific binding of secondary ab (use 10 % normal serum form the host animal of sec. ab; Goat-Anti-rabbit ==> normal Goat-serum). and 3. blocking of endogenious biotin with the Avidin/Biotin-blocking kit (Vector Labs). Use 0.2% Triton-X100 in PBS or TBS with 1 normal-Serum as delution medium for your antibodies. Make a delution raw of your primary ab to optimize the delution and run a negativ control (no primary ab) to get an impression which part of your reaction protocol is responsible for unspecific backgroundstaining.
Good luck!
Yes, the questions are: fixed or fresh sections, embedded or cryostat, cryoprotected or not a.s.o.
Anyhow, at 40 micros you have some neurons overlapping. This thickness is good for seeing well the architectonics, not the individual reacting cells. One section in a well is better, otherwise they stay one over the other and may also fold, which slows down the penetration of reagents and make unpredictable their amount per piece of the section.
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