Developing a protocol to IF label neuromuscular junctions on 20um thick formalin fixed, paraffin embedded sections captured on regular (+) slides however the muscle keeps falling off the slides. Samples are sectioned, dried, incubated overnight @ 60c, standard (organic) deparaffinization, antigen retrieved (Citrate pH6.0 with 0.01% Tween) 80C for 60 minutes, washed in DH2O permeabilized (pbs with 0.01% triton X) 80C 30 minutes followed by washing DH2O.
Tissues begin to come off after retrieval step and are completely lost after permeabilization. Have tried varying concentrations of detergents.
Any suggestions or guidance?
TIA
Hi Luis,
Loosing sections is always unpleasant!
As far as I can see, the incubation time at high temperatures for your antigen retrieval is quiet long. I made good experiences with microwave retrieval in citrate buffer. Therefore I "cooked" my slides in the microwave for 1min and let the cuvettes then cool down to roomtemperature. Another option would be to use sticky slides such as chromalaun gelatine or silane coated ones.
I hope I could help a bit.
I agree with Maria. I never fully understood the use of a microwave, but it has definitely worked for my samples in the past. Do you really need tritonX?? Could you coat your slides with an inert film or maybe use my old favourite (now sadly overlooked for preference expensive fancy new films lol) of a 1percent agar? Just a thought.
Hi Luis,
you could try serial (10 micron sections), which alway stick much better to the slides than 20 micron sections, and omit triton x.
Best regards, Daniela
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