Peggy, Have you tried the alcian blue tetrakis (methylpyridinium) chloride (Sigma, Cat. No.A4045, 90% dye content)?
I would be interested to know whether this stains the amyloid better. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) 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: [email protected] [mailto:[email protected]] On Behalf Of Lee Wenk Sent: Wednesday, 4 March 2009 7:35 AM To: Nathanial nauss; [email protected] Subject: Re: [Histonet] Sulfated Alcian Blue Sulfonate Alcian Blue (SAB) is not always as specific as you would like. Also, if you are using the newer alcian blue dye, it seems to have a different formulation than the old stuff that I have. The new alcian blue will not dissolve in alcohol, so the newer dye doesn't demonstrate the amyloid, while my old dye does. Old deposits of amyloid do not have beta pleats at regular intervals. Therefore, the Congo red dye (CR) will still bind, but will not line up one right after the other | | | | |, but will be more random \ _ | /. When the CR dye are parallel to each other, they will show the apple green birefringence with the polarizing microscope. When the CR dye is randomly arranged, there will be no apple green birefringence. The other time this happens is with very overfixed amyloid, such as months in NBF. Too many cross-links with NBF, so the CR dye can't bind right, so they are not in parallel. We also had this happen once when the autopsy tissues were fixed in B5 (mercury fixative, long time ago). The resident knew amyloid was an immunological problem, so put through tissue fixed in B5. Congo red did not birefringe. So we went back to the NBF stock bucket, submitted new tissue, and they all were wonderfully green birefringent. The other problem with the SAB is that, if this is old amyloid, and the beta pleats are messed up, SAB depends somewhat on the beta pleats. Therefore, the green will be much paler in older amyloid than with newer amyoid. (The green is due to to the blue of alcian blue staining the amyloid, and the yellow of picric acid in the van Gieson also staining the amyloid, so blue and yellow make green.) We just had a case of non-birefringing green Congo red at our hospital a couple of months ago, where it definitely looked by amyloid on the H&E, but there was no birefringence on the patient's CR (control was great - all 3 times that we repeated the procedure). Ot wasn't a fixation problem, or a staining problem, but probably an "old amyloid" problem. Our resident Dr. Tom Fennel just gave a talk on it at our state histology's winter seminar Jan. 31, 2009. Here's what we did (all three): 1. View the Congo Red stained slides with a fluorescence microscope, such as in microbiology auramine-rhodamine stain for AFB. When hit with green light, the CR stained amyloid will fluoresce orange. 2. Use Crystal Violet or Methyl violet staining for amyloid. These depend upon the carboxyl ions of the amyloid for binding, not the beta pleats. The amyloid should be violet, with the background blue/purple. However, it doesn't always demonstrate AA amyloid (some are low in surface carboxyl ions). So not always as sensitive as CR. 3. Use Thioflavin T or Thioflavin S for amyloid. Staining is (maybe) with the P component of amyloid, not the beta pleats. Use a fluorescence microsope, using blue light (FITC filters), and the amyloid will fluoresce yellow. However, other things also fluoresce yellow, such as fibrinoid material, JG granules, sometimes elastin, etc. So not as specific as CR. By knowing your histology and knowing where in the tissue you are thinking that there are amyloid deposits, the above three alternatives are a nice addition, for when Congo red is not demonstrating the apple green birefringence. Since in our recent case, 3 out of 4 stains demonstrated amyloid (CR fluorescent, Crystal violet and TFT were all positive, while CR birefringence was negative), it was diagnosed as amyloid, with, I think, a note that older deposits of amyloid sometimes demonstrate no birefringence with CR. If this person needs help with find staining procedures for CV/MV or TFT/TFS, could someone email them? I'm on vacation, and don't have access to my home or work computer. Peggy A. Wenk, HTL(ASCP)SLS Beaumont Hospital Royal Oak, MI 48073 ----- Original Message ----- From: "Nathanial nauss" <[email protected]> To: <[email protected]> Sent: Friday, February 27, 2009 10:42 AM Subject: [Histonet] Sulfated Alcian Blue I need some help, has any one used the sulfated alcian blue to stain for amyloid. We have a case that looks like it should be positive but it is not staining with the Congo Red. Any help would be great. Nathaniel _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************* 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. 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