I have rat brain sections (OCT) of 25 micron thickness. I am looking for microglia activation using IBA1 Monoclonal Antibody (GT10312), a known marker of microglia. I follow the general immunofluorescence protocol, including 1 hour of blocking followed by overnight primary antibody incubation at 4 degrees Celsius (concentration 1:100). The next day, incubation in the secondary antibody raised against the primary but not getting any signal. what could be the possible reasons behind this and what should do?
Did you do any permeabilization step? IBA1 is a cytosolic protein in microglia, so your antibody can only bind if the cell membranes are made permeable after fixation. Without permeabilization, the antibody can’t reach the target.
Alif Ismail Yes, I did permeabilization for 10 min, followed by blocking. I had even reduced blocking to 30 minutes, despite not getting any signal. I wonder if my antibody is suitable for frozen sections or not.
If you're using thermo ab, they should work. It is validated for for IHC on frozen sections based on their datasheet. What secondary antibody you used? It should be antimouse igG as per datasheet.
What's your permeabilization agent? Have you tried increasing the concentration? I think you could try increasing the permeabilization time also. 10 min might be insufficient for your 25micron tissue section. It's usually enough for
Alif Ismail Yes, the secondary antibody is anti-mouse. I am using 0.2% TritonX 100 (in 1x PBS). Sure, I will increase the time for permeabilization. Thank you for the help.
Priyanka Thakur you can try to keep the tritonX concentration at 0.2% and increase the permeabilization time to 15-20 minutee. Update me the result and goodluck!
How old is your sample and AB? I faced a similar problem because my samples were too old when i sectioned them. Try multiple concentrations of the primary AB. Increase the time for permeability like Ismail said. Good luck.
Nasar Ullah Khan Niazi, I received the AB in February, but MfD is not mentioned. My sample is 2-3 months old. Ok, I will increase the Ab concentration.
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