After sacrifice of animals, I am keeping pancreas in 4% paraformaldehyde for 1-2 weeks. Then for rehydration in 15% sucrose for 1 day before going for sectioning. I am using cryotome for making sections of pancreas embedded in OCT medium. Slides were kept in -80 degrees. After staining with H & E , I am not getting intact sections.
Since you are fixing tissues, instead of using a cryotome, why dont you try the routinely followed paraffin embedded tissues for obtaining the desired sections..
I agree with Vinay, if you are just doing H/E then why not paraffin, much easier than cryosections and studier. Also, 1-2 weeks in PFA is a long time, for whole tissue I generally do about 24-48 hours, wash very well, and then run through any general protocol for paraffin embedding. Also, how thick are your sections? If you are finding the sections are tearing or the sectioning is not going well, you can also increase the thickness of the sections, especially since you are only doing H/E
I'd like to suggest the use of Gendre Fluid (alcohol 95-96% with Picric Acid diluted until saturation+Acetic Acid+Formalin) (=Alcoholic Bouin) fixing by 3 days (maximum). Epithelial (glandular) tissues are very delicate and Gendre fluid will fix the tissue fast. This fixing will permit you to develop PAS protocol for glycogen too, and the tradictional H-E.
Literature: http://pt.slideshare.net/ahmededrissi1/routine-histological-techniques
Best regards.
I forgot to write that the microtome blader can be always changed because it can develop indentation. These indentations can cause cutting artifacts and the pancreas slides could appear with grooves on it.
Moreover, cryosections are did without fixing (in naturae) to maintain the histological biochemistry properties. I think it is important do some tests: cryosections without fixing, cutting with fixing in Gendre Fluid (or traditional Bouin) with inclusion on paraffin, cryosections with fixing in Gendre fluid (or traditional Bouin) and discuss your results on the paper and stablish a clear and truthworthy protocol to Rodentia pancreas.
I think 1-2 weeks duration in 4% PFA is not a big Problem if you are only using stainings like H&E, Trichromestains like Masson. An important fact is the cryoprotection. You have written you left the tissue just for 1 day in 15 % sucrose. I recommand to change it to 30 % sucrose leave the tissue until it sunks on the ground of your vial where you store it. A proper fixed , clean microtome blade or knife as Renato mentioned it is also essential for good sections. Play with the temperature of the block and the chamber. Somtimes when the termperature is to cold or when you cut to fast the sections could fray out . Do the sectioning slowly but continuously. If you have the possibility to do paraffin sections, this will be the better way, because you can get thinner sections with a better resolution under the microscope.
Hello dear, I already manipulated with pancreas tissue in mice for H&E staining, I used a routine stain which is fixing in 10%of buffered formaline, because you know that the pancreas contain so many enzymes, but the trouble shot may be your knife is so tilt, or the temperature of your block.
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