Hello -
I have been attempting to macrophages via IF with F4/80 in OCT-embedded mouse skeletal muscle using Thermo Rat Anti-Mouse monoclonal Ab (Cat# MF48000) with little success. I have been allowing sections to warm to RT for ~10 min, fixing for 15 min in 4% PFA at RT, followed by blocking for 1 hr at RT in 5% NGS in PBS-T, then O/N at 4C diluted 1:100 in primary Ab.
Has anyone had any success with this IF protocol? Do others use antigen retrieval?
Any advice would be appreciated!
Hi Olivere!
I have done IF for F4/80 on frozen mouse corneal section (4% PFA fixed). And, i got staining easily.
For this, i did following steps:
1. Frozen sections were kept at room temperature (RT) to thaw briefly.
2. The sections were washed with 1XPBS 2 times, 10 mins each. The slides were not allowed to dry completely from this point.
3. Next, sections were permeabilized with 0.1% Triton X-100 for 30 mins at RT and blocked with 10% goat serum for 1 hr at RT.
4. Slides were incubated overnight with primary antibodies at 4°C after draining the blocking buffer.
5. Next day, slides were incubated with secondary antibodies at 37°C for 1 hr.
So, there was no need for antigen retrieval step. You can also try to follow these steps for your tissue.
Hope this will help to solve your problem. Good luck...
Hi Lindsey,
Consider the following points:
1. Change your primary antibody from Rat anti-mouse to Rabbit monoclonal antibody
2. Have a look at abcam's website, they have several F4/80 antibodies.
3. Use anti-rabbit secondary antibody, preferably FITC or TRITC labeled
4. Introduce a permiabilization step as suggested above.
Best Regards,
Shafiq
If looking at the information sheet by Thermo, the antibody is described for use with either frozen + acetone-fixed or paraffin-embedded + PFA-fixed sections. I would try an acetone fixation of your frozen sections as already suggested by Gulab or try non-fixed sections. Don't forget a secondary-only control to make sure the signals are antigen-specific... By the way: You did not tell us which secondary antibody you use. It might also be a matter of its specificity.
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