Well said John - *eats grass and leaves speaks to the hand* AbuDhabiAnnie
2009/12/9 John Kiernan <jkier...@uwo.ca> > This is a typical example of the informal "protocols" that get passed on > from generation to generation of graduate students, postdocs and technicians > in research labs at universities. The original was probably written by > someone who knew how to do H&E staining, but on differently fixed tissues, > and certainly on thinner sections. It apears to be for individual slides, > because 10 seconds in each of the two 95% and 100% ethanols would be > effective only with vigorous agitation in a large excess of fluid. > > The tissue is almost unfixed, unless "Soak in 0.4% paraformaldehyde" means > leave it ovenight or longer in 4% formaldehyde. > Researchers otherwise educated to the highest levels in such difficult > disciplines as molecular biology and neuroscience regularly write phrases > like "4% paraformaldehyde", thereby advertising their profound ignorance > about fixation, which is the procedure that has the greatest effect on the > appearance of anything dead that's examined with a microscope, especially if > stains or histochemical methods are to be used. (I apologise for the length > of the preceding sentence, but not for its punctuation, which is correct in > British but not in American English usage. Check it out with Lynne Truss!) > > The "sucrose cycle" step, with no times or instructions about floating and > sinking, is probably local jargon from a lab where small animals' brains are > minimally fixed and cryoprotected before cutting thick (50-100um) frozen > sections, to be stained free-floating. That's not an H&E job! You are > working with a thin skeletal muscle (rat's gastrocnemius). > > If your Mayer's haemalum is a bought solution, it is intended for use in > hospital labs, on paraffin sections about 5um thick. In a research setting > you may need to make changes. Haemalum (Mayer's or anyone else's, correctly > used) should stain cell nuclei blue and very little else. > > An important part of H&E staining is looking at the wet section with a > microscope to check for adequate and selective nuclear coloration. In > skeletal muscle the nuclei are small, so the haemalum-stained section is > very pale blue to the unaided eye. With alcoholic eosin (as in your method) > it's not so easy to control the staining with microscopic control, but it > will probably be OK if the section is light pink. Some people like their > eosin darker; it's largely a matter of taste unless you need to distinguish > between different eosinophilic components on the basis of hue. > > John Kiernan > Anatomy, UWO > London, Canada > = = = > -----Original Message ----- > From: Josephine Garcia <j...@u.washington.edu> > Date: Monday, December 7, 2009 11:43 > Subject: [Histonet] Overstaining - Mayers H&E > To: histonet@lists.utsouthwestern.edu > > > Hi all, > > > > My (frozen-section, fixed) slides are coming out much too dark > > (overstainedpurple) and I'm not sure why. They are 15-20 > > micrometer slices of rat > > gastrocnemius muscle. Can someone please look over our current > > protocol and > > tell me what I'm doing wrong? Thanks! Here it is: > > > > 1. Perfuse animal with 4% paraformaldehyde fixative. > > 2. Soak in 0.4% paraformaldehyde > > 3. Sucrose cycle (5% rinse, 10%, 20%, 30% soak) > > 4. Embed in OCT, Frozen sections (15-20 micrometers) > > 5. Let dry for 15-30 min > > 6. Stain as follows: > > > > - Distilled H2O (quick dip) > > - Mayer's Hematoxylin - 1min (originally we were dipping for 5- > > 10 minutes. I > > slowly reduced the time to 2min, then 1min, then 30s... still > > overstained!)- Running lukewarm tap water - drain and > > continuously fill - 15min or until > > water runs clear > > - Distilled H2O (quick dip) > > - 80% EtOH - 1-2min > > - Eosin - 2 min > > - 95% EtOH I - 10sec > > - 95% EtOH II - 10sec > > - 100% EtOH I - 10sec > > - 100% EtOH II - 10sec > > - Xylene I - 2min > > - Xylene II - 2min > > > > 7. Coverslip and let dry overnight > > _______________________________________________ > > Histonet mailing list > > Histonet@lists.utsouthwestern.edu > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > -- Anne van Binsbergen (Hope) Abu Dhabi UAE _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet