We used breast carcinoma tissue, running IHC used Leica Bond Max. The detachment tissue that we found is about 30%. All instrument (eg slide, antibody ER, PR) we used is from Leica.
We don't understand about this high detachment percentage. We need your suggestion or sharing your experience using Bond Max..
Thank you..
Hi,
In paraffin sections I found it of paramount importance that the sections are properly dried before staining. So when the frozen slides were prepared for staining, they would need to be dried for several hours in a fume hood. Furthermore, I chose the coated + quality slides for my samples. This way I didn't have to apply any additional fixing protocol.
Hi, we have used Poly-Lysine and is really helpful:
http://www.sigmaaldrich.com/technical-documents/articles/biofiles/poly-lysine.html
Regards.
Dear Ruth,
I have done alot of immuno work during my masters and PhD. I used to clean and coat my own glass slides and never had detachment problem. It is relatively simple and cheap. Basically you clean your slides with ethanol and then dry them in the oven in slide racks. Then you coat them with gelatin or poly-lysine. Dry again and store them in a dust free environment-containers. For details you can have a look at my PhD thesis and see if you can extract some useful tips.
Best wishes,
Refik
Thesis Molecular physiology of ATP-gated ion channels assembled fro...
Hi,
In paraffin sections I found it of paramount importance that the sections are properly dried before staining. So when the frozen slides were prepared for staining, they would need to be dried for several hours in a fume hood. Furthermore, I chose the coated + quality slides for my samples. This way I didn't have to apply any additional fixing protocol.
Dear Ruth, mount your sections on Superfrost Plus slides. Lab ware companies sell them. If you are working with praffin sections try to cut them as thin as possible (3-5µm). Precooling is helpful as well. That meens put your tissue blocks and the microtome blades or knifes in the fridge before you start cutting. Dry the mounted sections several hours ore over night in the oven at 37°C. Good luck!
As director of a Morphology/Histology Research Core, where we immunostain many different tissues from various species (mouse, human, monkey, rabid, sheep), I would highly recommend that you let both your paraffin and frozen sections dry over night at room temperature before storing or using the next day. This is the most important step. However, for especially problematic tissues (such as cartilage), we use TruBond300 slides (which I think are coated with a seaweed extract). The only drawback is that these slides are so adhesive that you cannot refloat and reposition paraffin sections easily on the slide. Once you pick the sections up, they are there to stay!
So . . . you definitely want to use charged or coated slides and let the sections dry overnight.
I've used the Leica Bond Max (great machine) and tissue sections floating off has nothing to do with the immunostainer. Use plus charged slides, air dry sections at room temperature then dry in a 65C oven for an hour or so.
Fatty breast tissue is very temperamental. Difficult to fix and process and the sections tend to fall off slides.
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