Hi,
Before I used to use waterbath for antigen retrieval step (citrate buffer) for IHC, but now I am advised to use microwave. I performed IHC 2-3 times, but I observe partial staining or no staining. I exposed tissue sections maximum for 3 min when I used microwave. So, I feel that the problem is with the antigen retrieval step. I am working on paraffin embedded brain tissue sections. Please guide me through this issue.
Thanks,
Hi Amisha,
as the colleagues told, for pH6 up to 20 minutes.
But i would prefer to stay at water bath. you can get better consistent results. With the microwave, you got the Problem, that the heat for the pretreatment came in waves and is not continuous.
Put your cuvettes with the pretreatment buffer into the water bath and then starting the heat up.
After some minutes ( maybe 15 ) controll the heat in the cuvettes with a Thermometer. The heat must be something around 98°:
If you put then your slides into the heated solution of your cuvette, the temperature will fall down! Down to 92°, it's depend of the amount of your slides.
Then you Close your waterbath and measures the temperature again. If the temperature in the cuvette is again at 98° then you start your counter ( up to 20 minutes ).
After the time, remove fast the slides and wash them in cold wash buffer to decreased fast the temperature.
Just try it!
Cheers Robert
Hello Amisha,
Usually we do 15-20 min microwave in citrate buffer. However, you need to optimize the conditions for each antibody. So try with 15 and 20 min first and modify your protocol as per your needs.
With best
Hi Amisha
We adjust microwave timer at exactly 21 min for citrate buffer. Of course, try optimization for each antibody. Sections should boil at least 10 min. Most of our protocols suggest 10-20 minutes.
Best regards, Milica
Hi Amisha, I run 4 x 5 min microvawe at 800 w in 0.01 M citrate buffer pH 6 for 5 µm thick paraffin sections. I recommand to titrate the optimal antibody dilution because it depends on the detection system you use. Good luck!
Hi Amisha,
as the colleagues told, for pH6 up to 20 minutes.
But i would prefer to stay at water bath. you can get better consistent results. With the microwave, you got the Problem, that the heat for the pretreatment came in waves and is not continuous.
Put your cuvettes with the pretreatment buffer into the water bath and then starting the heat up.
After some minutes ( maybe 15 ) controll the heat in the cuvettes with a Thermometer. The heat must be something around 98°:
If you put then your slides into the heated solution of your cuvette, the temperature will fall down! Down to 92°, it's depend of the amount of your slides.
Then you Close your waterbath and measures the temperature again. If the temperature in the cuvette is again at 98° then you start your counter ( up to 20 minutes ).
After the time, remove fast the slides and wash them in cold wash buffer to decreased fast the temperature.
Just try it!
Cheers Robert
Hi. using microwave oven for antigen retrieval need to optimizing and may be try several times, but I think using proteinase K for this aim is efficient and providence in time.
I know it's an old question but I'll add some insights :-)
Very often people forget about "parameters," which is very important in research, very important for repeatability of assays. What is the temperature people achieve in the microwave regardless of Watts power? I am doing Opal staining and for one of the steps I was told: 1 min at 100%, 15 min at 10% (no Parameters? I asked what is goal temperature and I didn't get an answer). I troubleshooted my protocol and this is what I use with good results.
- Heat about 250 ml of tap water for 80 sec at 100% microwave power energy (MPE). I use an empty 1 ml tip box.
- Then I put in the hot water the jar with slides (without the jar's top), filled to top with antigen retrieval solution (ARS), and heat for 80 sec at 100% MPE. I use the tip box's lid to cover the box to help retain moisture, "not to seal."
- Then I heat again for 80 or more seconds until it boils. For this, I uncover the box to view it from outside the microwave, I stop immediately the moment it starts boiling (which will risk loosing the sections). Don't worry about the tap water boiling on the box, care about the bubbles on top of the ARS.
- Check the temperature in the ARS.
With this procedure I have been able to consistently reach about 90-92ºC (this is your parameter #1).
- Then I lower the MPE to 10% and heat for 15 min with the jar covered (do not seal or it risk to boil). This has allowed me to consistently reach about 75-78ºC at the end of the 15 min, without boiling or ARS evaporation (this is your parameter #2).
Check what are the parameters that best work with your target antigen (temperature + time + ARS).
Hope this will help you.
We microwave in citrate buffer (pH 6) for 10 minutes in three small time intervals (3 minutes + 3 minutes + 4 minutes) and it works just fine!
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