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Re: AW: [Histonet] Movat Pentachrome - Need to enhance Alcian Blue
Hi Gudrun How deep would you consider the oxidation to go? I have tried samples from around 200um into the block. The Alkaline Alcohol converts the alcian blue to monastral fast blue, which is insoluble - this is what i have been told. So locks it in I guess. Doesn't really matter as I don't get alcian blue in the sample to lock in. Thanks Krista On May 28, 2013 10:09 AM, Gudrun Lang gu.l...@gmx.at wrote: Hi Krista, Maybe the bad stainability is more due to oxidation by air of the old block-surface than due to fixation. A deeper slide for comparison would show. I don't know what the alkaline alcohol is good for ? Gudrun -Ursprüngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Krista Sider Gesendet: Montag, 27. Mai 2013 23:28 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] Movat Pentachrome - Need to enhance Alcian Blue Hi All, I had wonderful help from this site for my Woodstain Scarlet-Acid Fuchsin binding collagen problem. Now that this is fixed I have realized that the Alcian Blue is not staining my excessively fixed samples (Human Aorta Aortic Valve, formalin fixed 2-4 years, embedded in paraffin and stored at RT for ~6 years). I have some samples fixed for only 2-6 months and these are fine but the samples fixed for 2-4 years do not uptake the Alcian Blue. I have taken it up to 1 hr in the 1% Alcian Blue solution followed by 10 min in 56*C Alkaline Alcohol solution. I do a 1hr Bouin's 50*C to start (I have also tried Bouin's at 60*C). Any ideas on how I can get the Alcian Blue to stain these excessively fixed samples or possibly a substitute? I need to see the proteoglycans/GAGs. Thank you Krista ___ 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
[Histonet] Movat Pentachrome - Need to enhance Alcian Blue
Hi All, I had wonderful help from this site for my Woodstain Scarlet-Acid Fuchsin binding collagen problem. Now that this is fixed I have realized that the Alcian Blue is not staining my excessively fixed samples (Human Aorta Aortic Valve, formalin fixed 2-4 years, embedded in paraffin and stored at RT for ~6 years). I have some samples fixed for only 2-6 months and these are fine but the samples fixed for 2-4 years do not uptake the Alcian Blue. I have taken it up to 1 hr in the 1% Alcian Blue solution followed by 10 min in 56*C Alkaline Alcohol solution. I do a 1hr Bouin's 50*C to start (I have also tried Bouin's at 60*C). Any ideas on how I can get the Alcian Blue to stain these excessively fixed samples or possibly a substitute? I need to see the proteoglycans/GAGs. Thank you Krista ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Movat Pentachrome - Can't Remove Woodstain Scarlet-Acid Fuchsin from Collagen
Thank you all very much for you help. I really appreciate it. Gudrun: I tried the PTA before the WSAF and it worked beautifully (1 min PTA, dH2O, 5 min WSAF, dH20, AA, 10 min PTA). Red muscle and clean collagen.Thank-you Liz: I didn't get to trying the 60*C Bouin's but I will make a note for next time I am optimizing. Thanks again Krista On Sat, May 11, 2013 at 4:04 AM, Gudrun Lang gu.l...@gmx.at wrote: Try to stain first in PTA/PMA solution to impregnate the collagen fibers - perhaps testing with different times. Then follow up with red stain and the usual procedure. We use a stain called SFOG, that first impregnates 2 min with PMA and afterwards with the mixture of Chromotrop, Acidfuchsin and Anilinblue for 10 min. The longer we do the Polyacid-step the more intensive are the fibres and less intensive is the cytoplasma. I think, if after this trial the collagen is still red, that there are binding-sites in the collagen, that can't be influenced. Maybe acidfuchsin is here the main partner and a pure solution of Scarlet Red may help. Gudrun Lang -Ursprüngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Krista Sider Gesendet: Freitag, 10. Mai 2013 23:13 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] Movat Pentachrome - Can’t Remove Woodstain Scarlet-Acid Fuchsin from Collagen Hello All, I have successfully stained porcine and mouse paraffin embedded heart tissues with Movat’s Pentachrome (MP), using EMS’ MP solutions and protocol (http://www.emsdiasum.com/microscopy/technical/datasheet/26385.aspx), with some optimization on times. However, now I am working with archival human tissue that has been fixed for much longer and is much older than my other samples (Human Aorta Aortic Valve, formalin fixed 2-6 months or 2-4 years, embedded in paraffin and stored at RT for ~6 years). I cannot seem to get the Woodstain Scarlet-Acid Fuchsin (WSAF) to pull out of the collagen with the 5% Phosphotungstic Acid (PA) or 1% Phosphomolybdic acid (PM) Acid. I have tried increasing the Bouin’s initial fixation (max 2 hrs 50*C), diluting the standard WSAF solution by 50% (1 dip) and taking the PA or PM up to 1 hr, yet there is always a lot of red still left in the collagen, in some regions as strong as the muscle (which I am sure are not muscle or dense cells). I see virtually no change with increased PA or PM time. As I have aorta in my samples I can’t just leave out the muscle stain. I would be very grateful for your insights into anything I could try to get clean collagen and stained muscle in the MP stain. Why might my method not be working? Is there something I can substitute for the WSAF that might be appropriate? Thank you very much for you help, Krista ___ 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
[Histonet] Movat Pentachrome - Can’t Remove Woodstain Scarlet-Acid Fuchsin from Collagen
Hello All, I have successfully stained porcine and mouse paraffin embedded heart tissues with Movat’s Pentachrome (MP), using EMS’ MP solutions and protocol (http://www.emsdiasum.com/microscopy/technical/datasheet/26385.aspx), with some optimization on times. However, now I am working with archival human tissue that has been fixed for much longer and is much older than my other samples (Human Aorta Aortic Valve, formalin fixed 2-6 months or 2-4 years, embedded in paraffin and stored at RT for ~6 years). I cannot seem to get the Woodstain Scarlet-Acid Fuchsin (WSAF) to pull out of the collagen with the 5% Phosphotungstic Acid (PA) or 1% Phosphomolybdic acid (PM) Acid. I have tried increasing the Bouin’s initial fixation (max 2 hrs 50*C), diluting the standard WSAF solution by 50% (1 dip) and taking the PA or PM up to 1 hr, yet there is always a lot of red still left in the collagen, in some regions as strong as the muscle (which I am sure are not muscle or dense cells). I see virtually no change with increased PA or PM time. As I have aorta in my samples I can’t just leave out the muscle stain. I would be very grateful for your insights into anything I could try to get clean collagen and stained muscle in the MP stain. Why might my method not be working? Is there something I can substitute for the WSAF that might be appropriate? Thank you very much for you help, Krista ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
