[Histonet] re:Hematoxylin Staining of Skin
My first question is in what region of skin is the staining poor? The fibroblasts of the dermis should stain really well with HE = as should the cells of the stratum germinativum. From there out nucle ar staining gets worse and worse. I attribute it to two things, and i= f anyone else would add I would love more insight into this problem: = First is keratinization. Since cells are no longer dividing and are g= radually dying and losing nuclei as they make their way out to the stratum = corneum, nuclear staining is going to be sparse as a result of the process = itself. Also, highly keratinized epithelia tend to be problematic for= many embedding and staining techniques where tissue has to be dehydrated a= nd rehydrated. I have always had trouble sectioning highly keratinize= d skin because of improper infiltration. Second is the organization o f chromatin in epidermal cells in general, which is seemingly very differen= t than other epithelial cells (again, the stratum germinativum often does s= how typical nuclear staining). With all that being said, I hav= e tried several things to remedy problems associated with HE staining = of skin with mild success. Sometimes it might be as simple as adding = a few drops of glacial acetic acid to your hematoxylin to lower the pH.nbs= p; When this doesn't work, I typically will use iron alum or ferric chlorid= e (I believe 2-4% iron alum has worked relatively well for me when used) as= a mordant prior to staining with Delafields or Ehrlich's hematoxylin. = ; Destain with 2-4% iron alum or ferric chloride because anything holding t= he alum when you stain picks up hematoxylin and will need to be differentia= ted before counterstaining. I have also had mild success with iron ga= llein elastin stain, but this is really more of a stain for elastin that ju st happensto stain nuclei blue-black and gives good differentiation. = Gallein is also not as easy to find as Hematoxylin. As I said= , if anyone has information on either the chemistry behind this or maybe ev= en on the organization of chromatin in epidermal cells, the histo community= is all ears. -Matt ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re: staining of elastin fibers (resend)
This is a resend of a previous message that seems to have been chopped to pieces in cyberspace. Hope this one comes through better: I prefer to use iron gallein elastin stain for demonstrating elasti fibers. It is simple to make up and gives good contrast compared to some of the other stains. Nuclei stain dark blue, elastin fibers black, everything else is pink or light purple. The protocol can be found in Humanson's Animal tissue techniques and the original article is by Churukian and Schenk (Stain Tech., 1976). I can provide a protocol in .doc format I can send, along with any notes, if you don't have access to either source. Current suppliers of gallein are Fisher Scientific, Spectrum Chemicals, and ArtChemicals.com. Spectrum has it cheapest. Good luck. -Matt - Matthew T. Close Lehigh University Department of Biological Sciences ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re: staining of elastic fibers
Weihua, I prefer iron gallein elastin stain because it is = very simple to make and use and gives good contrast compared with some of t= he other techniques. The recipes and protocol can be found in Humanson's An imal Tissue Techniques (in addition to many other histological techniques t= exts), but I can also supply my protocol if need be (please email and I wil= l send an attachment). Elastic fibers stain black with gallein, nucle= i pick up some of the stain and will be dark blue-black. Differentiat= ion is done with ferric chloride and the counterstain is your personal choi= ce (although acid fuchsin in saturated picric acid is typically used with g= ood results). The gallein itself is hardest to find, but can currently be p= urchased from Fisher Scientific, Spectrum Chemical, or ArtChemicals.com.nb sp; I believe that spectrum has it cheapest, and I would buy at least 25g i= f you can because it sometimes goes off the market. Sincerament= e, Matt Close ---= -- Matthew T. Close Lehigh University Department of Biological Sciences ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] muscle skinning solution for fluorescence microscopy
I am in need of a good, working chemical skinnig technique for skeletal muscle fibers. I will be staining skinned, isolated fibrils with Rh-Phalloidin and DAPI stains for observation and analysis using a fluorescence scope. Thanks. - Matthew T. Close Lehigh University Department of Biological Sciences ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
