Hi there! Does anyone have any experience using the mouse monoclonal TMEM119 antibody from Proteintech (66948-1-Ig) for immunofluorescence? I have been trying to use it with paraffin-embedded rat brain tissue from animals at P28, but have had poor staining results (high background fluorescence, individual cells not distinct; see attached images). Using it at 1:250 and 1:200 concentrations delivers the same results. I was wondering if there is anything I should be doing in my protocol to improve my staining.
Currently, my protocol includes:
- Antigen retrieval with 3-min boiling in citrate buffer (pH 6) in microwave
- Permeabilisation of tissue by blocking in 10% NDS/0.1% Ttriton-X-100/0.1%Tween for 1 hr at room temperature
Any help would be greatly appreciated!
Hi...you could try out few conditions
1. Trying higher dilutions ( 1:400 or 1:500 of Pr-Ab) and secondary at 1:700 (Try to use Sec-Ab conjugated to 647 , perhaps better than 488 channel as at times autofluorescence at 488 can interfere with positive signal)
2. Perhaps include Tris-EDTA buffer for antigen retrieval
3. Try to include negative controls for each kind of new condition tried
Good luck.
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