Re: [Histonet] Amylase Digestion for glycogen
We also had problems with diastase digestion. We researched amylase and found it to be REALLY expensive. We found a reasonably priced source with American Master Tech. (www.americanmastertech.com/store/main.aspx?p=ItemDetailStylesitem=AHDIA50 ). We tried it this semester and the students obtained really good results, some better than the dreaded saliva. I have the PDF for the procedure if you would like it. Jennifer MacDonald Mt. San Antonio College Peterson, Dan 1dpeter...@meriter.com Sent by: histonet-boun...@lists.utsouthwestern.edu 10/03/2012 11:44 AM To 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu cc Subject [Histonet] Amylase Digestion for glycogen Amy, I was wondering how you made out with Amylase vs. diastase for your glycogen digestion. Can you send me your protocol if it worked out? We're all of a sudden not getting our malt diastase to work. Thanks Dan Daniel R Peterson HT(ASCP) Histopathology Section Head Meriter Laboratories (608) 417-6557 fax (608) 417-6343 1dpeter...@meriter.com CONFIDENTIALITY NOTICE: This message (including any attachments) is intended for the sole use of the individual and entity to whom it is addressed. This message may contain information that is confidential and is protected by law. If you are not the intended recipient, you are hereby notified that any disclosure, copying or distribution of this message is strictly prohibited. If you received this message in error, please immediately notify the sender by reply email and then delete the message. Thank you. ___ 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
RE: [Histonet] Amylase Digestion for glycogen
Amysue, Our amylase digestion procedure is quite simple (see: Mangan, V-M, Farago, V., Kelly, M., Henwood, A.F., (2002) An Amylase Reagent with a Long Shelf Life for the removal of Glycogen from Tissue Sections J. Histotechnol 25:153.) See below for the method: Amylase Reagent Warning: Harmful, contains azide - see MSDS Alpha Amylase from Bacillus Subtilis (Fluka Cat No 10070,) 1g Oxoid PBS Tablets (Cat No BR14a)1 tablet Distilled water 100ml Sodium Azide0.1g This solution, once prepared is stored at 4oC when not in use. A recycled antibody dropper bottle (often used in commercial immunoperoxidase kits) is useful for storage and application. Procedure: 1. Dewax and hydrate paraffin sections, hydrate frozen sections. 2. For amylase digestion, place slides on a rack, cover sections with amylase solution and allow to incubate for 10 minutes at room temperature. 3. Wash slides well in water. 4. Place slides in 1% periodic acid 10 minutes. 5. Wash slides well in water. 6. Rinse slides in distilled water. 7. Place in Schiff's reagent 10 minutes. 8. Rinse slides in distilled water and then wash slides in tap water 3 minutes. 9. Counterstain slides with haematoxylin, differentiate and blue. 10. Dehydrate, clear and mount. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Ruppert, Amysue Sent: Saturday, 5 February 2011 11:03 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Amylase Digestion for glycogen Hello, We are looking to switch from malt diastase digestion for glycogen to Amylase digestion. I have the new protocol worked up, but one of the Pathologists I work with would like to have an idea of how many labs out there are using Amylase instead of malt diastase for their PAS/D method. If you use amylase for the PAS/D method, could you please let me know who you are and the institiution? Much appreciated. amysue ruppert Histology lab Marshfield Labs Marshfield WI __ The contents of this message may contain private, protected and/or privileged information. If you received this message in error, you should destroy the e-mail message and any attachments or copies, and you are prohibited from retaining, distributing, disclosing or using any information contained within. Please contact the sender and advise of the erroneous delivery by return e-mail or telephone. Thank you for your cooperation. * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Amylase Digestion for glycogen
You are dealing with synonyms of the same enzyme but the amylase protocol always is slightly more complex. Stick with diastase and, please, never use your own saliva for this procedure René J. --- On Fri, 2/4/11, Ruppert, Amysue ruppert.amy...@marshfieldclinic.org wrote: From: Ruppert, Amysue ruppert.amy...@marshfieldclinic.org Subject: [Histonet] Amylase Digestion for glycogen To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date: Friday, February 4, 2011, 7:03 PM Hello, We are looking to switch from malt diastase digestion for glycogen to Amylase digestion. I have the new protocol worked up, but one of the Pathologists I work with would like to have an idea of how many labs out there are using Amylase instead of malt diastase for their PAS/D method. If you use amylase for the PAS/D method, could you please let me know who you are and the institiution? Much appreciated. amysue ruppert Histology lab Marshfield Labs Marshfield WI __ The contents of this message may contain private, protected and/or privileged information. If you received this message in error, you should destroy the e-mail message and any attachments or copies, and you are prohibited from retaining, distributing, disclosing or using any information contained within. Please contact the sender and advise of the erroneous delivery by return e-mail or telephone. Thank you for your cooperation. -Inline Attachment Follows- ___ 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] Amylase Digestion for glycogen
Hello, We are looking to switch from malt diastase digestion for glycogen to Amylase digestion. I have the new protocol worked up, but one of the Pathologists I work with would like to have an idea of how many labs out there are using Amylase instead of malt diastase for their PAS/D method. If you use amylase for the PAS/D method, could you please let me know who you are and the institiution? Much appreciated. amysue ruppert Histology lab Marshfield Labs Marshfield WI __ The contents of this message may contain private, protected and/or privileged information. If you received this message in error, you should destroy the e-mail message and any attachments or copies, and you are prohibited from retaining, distributing, disclosing or using any information contained within. Please contact the sender and advise of the erroneous delivery by return e-mail or telephone. Thank you for your cooperation. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Amylase Digestion for glycogen
div style=font-family: 'times new roman'; font-size: 1= 6pxdivIt is good that the pathologist you work with takes an= interest in histochemical methodology, but when and where did he lear= nelementarybiology?/divdiv br//divdivW= hen I was a schoolboy in the 1950s the biology teacher told us that dias= tase and amylase were different names for the enzyme in saliva and pancr= eatic juice that digests starch. This was also true in my two prec= linical years at medical school (early 1960s). The biochemistry teache= rs told us more about starch and its animal equivalent glycogen, both digestible by amylase. Our early-1960s textbooks told us about Claude = Bernard, the great French physiologist who discovered the properties= of glycogen in the mid-1800s and experimentally demonstrated the func= tion of the liver in glucose metabolism and homeostasis./divd= iv br//divdivAccording to the Sigma catalogue, di astase is now quot;an obsolete synonymfor alpha-amylasequot;.= The same catalogue lists alpha-amylases from many sources, includin= g malt. (They also sell beta-amylases, which would also catalyze = the hydrolysis of glycogen.) Sigma's least expensive alpha-amyl= ase is from human saliva./divdiv br//divdiv= Any amylase will do the job. Go with the cheapest. Human drooli= ngs are free and do not contain enzymes that will digest and solubilize polysaaccharides other than glygogen and starch. This is traditional h= istochemistry, well documented in textbooks and peer-reviewed papers f= or about 100 years./divdiv br//divdivJoh= n Kiernan/divdivAnatomy, UWO/divdivLondon, C= anada/divdiv= = =br /On 04/02/11, b class== namequot;Ruppert, Amysuequot; /blt;ruppe=rt.amy...@marshfieldclinic.orggt; wrote:/divblockq= uote cite=mid:201102050003.p15030nh007461@spamfilt class= iwcQuote style=border-left:#00f 1px solid; padding-lef= t: 13px; margin-left: 0px type=citediv class== mimepart text plainHello,br / We are looking to switch f= rom malt diastase digestion for glycogen to Amylase digestion. I have = the new protocol worked up, but one of the Pathologists I work with wo uld like to have an idea of how many labs out there are using Amylase in= stead of malt diastase for their PAS/D method.br / If you use = amylase for the PAS/D method, could you please let me know whoyouare=andtheinstitiution?br / Much appreciated.br /br = /amysue ruppertbr /Histology labbr /Marshfield Labsbr = /Marshfield WIbr /br / ___ 5F ___ 5F= __br /The contents of this message may= contain private, protected and/or privileged information. If you= received this message in error, you should destroy the e-mail message= and any attachments or copies, and you are prohibited from retaining,= distributing, disclosing or using any information contained within.= Please contact the sender and advise of the erroneous delivery by re= turn e-mail or telephone. Thank you for your cooperation.br / /div/blockquote/div ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
