Hi Meckenzie,

if you see the air bubbles after H2O2-treatment you can reduce the H2O2 concentration to 0,3% (30% H2O2 1:100) and do it in the fridge (4°C) for 20 min.

The reason for this bubbles are the reacton of endogene peroxidase. Peroxidase reduces H2O2 to H2O and O2. You see oxigene bubbles under your ssectons. If you use ascended or descended alcohols you can integrate this in an alcohol row (30% H2O2 in 100% ETOH at 4°C for 20 min).

If you are not using peroxidase labeled secondary antibodies or detections systems like ABC or strptavidin it is not nessary to do the H2O2 blocking step.

Hy,

when using fixed/frozen brains you can think about to perform your immunos with free floating sections (30 µm thickness). Thereby you have the advantage that your antbodies can penetrate from both sides into your slides.

Moreover, as Mohamed Mahdy mentioned you should put your tissue slides mounted on the super frost Plus slides for about 15 min on a heat plate at 37° C, so that they adhere correctly. Perhaps then you will have less air bubbles?

Hope I could help you. Kind regards

Stefan

Hello

You need to warm the slide up by using your finger. This step will help to mount the tissue on the slide.

Hello , Perhaps you need cryopreservation samples of tissue prior cryosectioning. In our lab we use special cryomolds (www.sealnfreeze.com). We can control bubbles in OCT and we have perfect slide all time.