[Histonet] ATPase 9.4 Troubleshooting
Dear Friends, I am having trouble with the ATPase 9.4 stain on muscle biopsies performed on frozen sections. The sections look extremely dark and almost have a digested look to them. To troubleshoot we: * made all new solutions, * opened a new container of ATPase (stored in the -15 deg. C. freezer) * the oven has been tested and confirmed to be stable at 40 deg. C. * the pH is stable at 9.4 (+/- 0.02) * only distilled water is used for all solutions where required and for rinses between steps. The ATPase 4.3 has some artifact but definitely is readable and not like the 9.4. All of the other sections for the other stains in the protocol (HE, Trichrome, NADH, ORO, PAS) are coming out great and the sections do not show any freezing or microtomy artifacts. Has anyone else run into this problem? We have run the same muscle cases 3 times in the last 2 days after making the changes to all of the solutions to prepare the ATPase. What are we missing? I may be able to upload a photo if you need to see the issue clearly. Thank you so much for your help! Karen Karen P. Bryan, HT (ASCP)cm Sr. Histology Specialist Department of Pathology/Amory 3 Brigham Women's Hospital 75 Francis Street/Boston 617-732-5454 (Internal extension 2-5454) The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: Masson Trichrome stain
Just out of curiosity--what strength of Picric acid is in your Bouins fixative?Nancy On Thursday, March 12, 2015 2:02 PM, Gayle Callis gayle.cal...@bresnan.net wrote: I have been following the string of inquiries about using metal forceps with Masson's Trichrome staining. I was taught many years ago to avoid metal forceps or the older metal tissue cassettes with Bouins. I am scrambling to find the actual reference. The reason given was acids in Bouins corrode metal. This may be a lost bit of information since the overall majority of labs now use plastic tissue cassettes. Case in point: using acidic descaling solutions for household cleaning i.e. showers/tubs or coffee machines. These solutions come with warning to avoid metal fixtures and stainless steel sinks. Accidental contact of acids in a stainless sink causes the metal discolor, indicating corrosion - been there, done that to a stainless steel sink. I so use metal forceps to move slides between Mass Tri staining solutions (and silver staining solutions) without problems per John Kiernan's comment. Not using metal forceps with silver stains i.e. GMS, reticulin, is to avoid metal ion contamination which is more likely due to with poorly washed glassware. In the past, we dipped metal forceps in melted paraffin, very messy since paraffin comes off on slides and in hot staining solutions. Disposable plastic forceps are cheap but break easily resulting in a dropped slide. Teflon forceps are pricey but it was a challenge to hold slides. Hopefully there are teflon forceps that work better than the one we used? We tried a teflon tipped metal forceps but not worth the price as teflon wears off the tips to rexpose metal. Weigerts hematoxylin is not affected by metal forceps since there are no acid components to corrode the metal although Weigerts can stain the forceps. Simply wash the forceps in dilute chlorine bleach then soap and water. I agree with John Kiernan and now use metal forceps to move slides between staining solutions in both Massons trichrome (and silver methods) without problems. If people want to use plastic or teflon forceps, I understand the reasons. As for not rinsing before going into Aniline Blue (or light green) in Massons trichrome, there is a reason for this. Sheehan and Hrapchak state verbatim The phosphomolybdic acid and phosphotungstic acid thus acts as a link connecting basic groups of the connective tissue fiber to the basic groups of the dye i.e. aniline blue. The PM/PT acid treatment has the ultimate effect of making an amphoteric dye that would ordinarily act as an acid dye to change and act as a basic dye. These authors also say Although the exact mechanism of how the stain works is unknown, some theories are available. By rinsing away the PT/PM, the link may be weaker hence one goes from PT/PM directly into aniline blue (sometimes light green or fast green). Bierbrich Scarlet/acid fuchsin and aniline blue (light green or fast green) solutions can be filtered back and reused many times. PT/PM and 1% acetic acid solutions should be discarded after use. Instead of kits due to expense and some kit deviations from classic Massons Trichrome method, I found I could buy excellent, reliable single staining solutions i.e. Biebrich Scarlet/Acid Fuchsin and Aniline Blue from Newcomer Supply or Poly Scientific to avoid exposure when weighing out carcinogenic dyes. Bouins is purchased from the vendor with the best price. However, PT/PM and acetic acid single use solutions were still made in house to save costs. I strongly recommend reading John Kiernan's Methods for Connective Tissues from his book , Histological and Histochemical Methods Theory and Practice for better explanation and understanding of Massons Trichrome chemistry. Collagen and muscle staining methods in Sheehan and Hrapchaks Theory and Practice of Histotechnology is not recent but a good start. Whew, a long reply but hope helps... Gayle Callis HTL/HT/MT(ASCP) Written is: Justine, I do not have any metal forceps in the special stains area, due to the reaction that they can cause when staining with silver. As a rule of thumb, it is just easier to use plastic all the way around. The Carson text does not state the use of only plastic forceps, but I would think that maybe they are concerned with a reaction between the Weigert's and the metal. That would be a stretch. As for no water before aniline blue, I believe the concentration is very weak and the water may dilute they dye even further. This would affect the staining results. Sincerely, Toysha N. Mayer, D.H.Sc., MBA, HT (ASCP) Instructor/Education Coordinator Program in Histotechnology School of Health Professions UT M.D. Anderson Cancer Center 713.563-3481 -- Message: 4 Date: Tue, 10 Mar 2015 00:31:56 -0500 From: John Kiernan
RE: [Histonet] RE: Masson Trichrome stain
I have always bought saturated Picric Acid and not a specific strength. Avoid making it from scratch!! Pam -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of nancy lowen Sent: Friday, March 13, 2015 3:57 PM To: gayle.cal...@bresnan.net; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] RE: Masson Trichrome stain Just out of curiosity--what strength of Picric acid is in your Bouins fixative?Nancy On Thursday, March 12, 2015 2:02 PM, Gayle Callis gayle.cal...@bresnan.net wrote: I have been following the string of inquiries about using metal forceps with Masson's Trichrome staining. I was taught many years ago to avoid metal forceps or the older metal tissue cassettes with Bouins. I am scrambling to find the actual reference. The reason given was acids in Bouins corrode metal. This may be a lost bit of information since the overall majority of labs now use plastic tissue cassettes. Case in point: using acidic descaling solutions for household cleaning i.e. showers/tubs or coffee machines. These solutions come with warning to avoid metal fixtures and stainless steel sinks. Accidental contact of acids in a stainless sink causes the metal discolor, indicating corrosion - been there, done that to a stainless steel sink. I so use metal forceps to move slides between Mass Tri staining solutions (and silver staining solutions) without problems per John Kiernan's comment. Not using metal forceps with silver stains i.e. GMS, reticulin, is to avoid metal ion contamination which is more likely due to with poorly washed glassware. In the past, we dipped metal forceps in melted paraffin, very messy since paraffin comes off on slides and in hot staining solutions. Disposable plastic forceps are cheap but break easily resulting in a dropped slide. Teflon forceps are pricey but it was a challenge to hold slides. Hopefully there are teflon forceps that work better than the one we used? We tried a teflon tipped metal forceps but not worth the price as teflon wears off the tips to rexpose metal. Weigerts hematoxylin is not affected by metal forceps since there are no acid components to corrode the metal although Weigerts can stain the forceps. Simply wash the forceps in dilute chlorine bleach then soap and water. I agree with John Kiernan and now use metal forceps to move slides between staining solutions in both Massons trichrome (and silver methods) without problems. If people want to use plastic or teflon forceps, I understand the reasons. As for not rinsing before going into Aniline Blue (or light green) in Massons trichrome, there is a reason for this. Sheehan and Hrapchak state verbatim The phosphomolybdic acid and phosphotungstic acid thus acts as a link connecting basic groups of the connective tissue fiber to the basic groups of the dye i.e. aniline blue. The PM/PT acid treatment has the ultimate effect of making an amphoteric dye that would ordinarily act as an acid dye to change and act as a basic dye. These authors also say Although the exact mechanism of how the stain works is unknown, some theories are available. By rinsing away the PT/PM, the link may be weaker hence one goes from PT/PM directly into aniline blue (sometimes light green or fast green). Bierbrich Scarlet/acid fuchsin and aniline blue (light green or fast green) solutions can be filtered back and reused many times. PT/PM and 1% acetic acid solutions should be discarded after use. Instead of kits due to expense and some kit deviations from classic Massons Trichrome method, I found I could buy excellent, reliable single staining solutions i.e. Biebrich Scarlet/Acid Fuchsin and Aniline Blue from Newcomer Supply or Poly Scientific to avoid exposure when weighing out carcinogenic dyes. Bouins is purchased from the vendor with the best price. However, PT/PM and acetic acid single use solutions were still made in house to save costs. I strongly recommend reading John Kiernan's Methods for Connective Tissues from his book , Histological and Histochemical Methods Theory and Practice for better explanation and understanding of Massons Trichrome chemistry. Collagen and muscle staining methods in Sheehan and Hrapchaks Theory and Practice of Histotechnology is not recent but a good start. Whew, a long reply but hope helps... Gayle Callis HTL/HT/MT(ASCP) Written is: Justine, I do not have any metal forceps in the special stains area, due to the reaction that they can cause when staining with silver. As a rule of thumb, it is just easier to use plastic all the way around. The Carson text does not state the use of only plastic forceps, but I would think that maybe they are concerned with a reaction between the Weigert's and the metal. That would be a stretch.
[Histonet] Medite Equipment
RE: Medite Equipment I was wondering if anyone has any experience with the Medite ACS720 Coverslipper or TST44 Stainer? We are thinking of purchasing one and would like to get opinions on the quality. Thank you Julie ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] (no subject)
please unsubscribe Live with Love, Joy and Vibrant Health ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
