As it is obvious that the level of TNF-alpha is increased following cerebral ischemia. I am going to perform the immunostaining for TNF-alpha positive cells in mice brain sections which is challenged with MCAo induced focal cerebral ischemia. I am going to use the DAB substrate for this purpose. Could anybody provide me the detailed protocol for this purpose?
I'm sorry. Unfortunatelly, I have never stained MCAO model mice withTNF-alpha antibody. and I don't have the antibody neither.
Hello I have done it to the spleen, and i have to confess that it does not look great with DAB9 (IHC), It looks better if you use IF: Here it is the protocol:
Wash 3X with TBS ( tris buffer saline) ph: 7.4
Incubate in SSC ( sodium citrate)(1 Molar concentration in TBS), ph :6 for 40 min at 80 Celsius.
Remove section from water bath, and let cool for 5 min.
Wash 3 X with TBS
Incubate in block for 60 mins ( TBS-TS) 0.1% tx-100 + 10 % goat serum
incubate in primary PBS-TS + primary ( 0.1% tx-100 + 3% goat serum + 1:50 Rabbit anti-rat TNF-α (NOVUS NBP1-19532) over night at 4 C
Day 2
wash 3X with PBS-TS ( 0.1% tx-100 + 3% goat serum)
Secondary Ab: incubate in 2Ab for 2 hours ( anti-rabbit from goat jackson cat# 111-065-144 for DAB staining ) or any Alexa fluoro anti rabbit secondary for IF.
For DAB: wash 3 x with TBS, then incubate in ABC ( Abivin complex) solution for 1 hour ( 2 drops of A and 2 drops of B).
wash 3X with TBS, and add DAB, then wash 3 X with TBS, then mount onto slide.
Ford IF: after alexa secondary wash 3X with TBS, and mount onto slide using vecta shield wet mount with dapi.
I hope this can help. kindly let me know the look of your staining.
Best wishes
sandra
Dear Sandra
Thank you very much for your detailed protocol. I will once try to do according to your suggestion and let you know as well.
One more question? Does the TNF-alpha positive cells in spleen appear as star shaped one or normal round one? I would be very happy if you provide me one photographs (or article) that you have regarding the TNF-alpha positive cells.
Again thanks a lot.
Dear Christopher Daniel Duntsch
I need the immunostaining methods. I have no problem for ischemic model, section methods or tissue preparation, and the antibody as well. Just wish to know the procedures for the TNF-alpha staining using DAB substrate.
Follow this link
http://www.ihcworld.com/_protocols/antibody_protocols/tnf-a_endogen.htm
Hello Christopher Daniel Duntsch
I like to know the IHC protocol for the TNF-alpha staining using DAB substrate.
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