Good day John, Very nice to hear from you again! I have been consulting your textbook in my investigations! Sorry about the brevity of the description of our method. I felt like my post was already too long and it might scare off some would-be contributors! :-) And yes, I incorrectly referred to the dichroic green as "fluorescent"-thank you.
Our method follows the Puchtler method described on pages 132-3 in Frieda Carson's "Self-Instructional" textbook (1990) as does the hospital that repeated our false-positive Congo Reds. Note, once we re-made our reagents, our results returned to accurate staining. Greg On Thu, Jun 20, 2024 at 2:49 AM John Kiernan <jkier...@uwo.ca> wrote: > Greg, your method is incompletely described in your Histonet post, but it > looks quite different from the "traditional" Highman's procedure (*Arch. > Path*. *41*:559-562). What method were they using "at another > lab" to get correct red amyloid that is green (dichroic, not fluorescent) > with crossed polars? > *John Kiernan* > > https://www.schulich.uwo.ca/anatomy//people/faculty/emeriti/kiernan_john.html > = = = > ------------------------------ > *From:* Greg Dobbin via Histonet <histonet@lists.utsouthwestern.edu> > *Sent:* June 19, 2024 8:53 AM > *To:* histonet@lists.utsouthwestern.edu <histonet@lists.utsouthwestern.edu > > > *Subject:* [Histonet] Causes of false positive Congo Red > > Hello experts, > *Some background:* > I know that Congo Red can bind nonspecifically to non-amyloid components > such as collagen and elastin under certain conditions (eg Carnoys fixative, > insufficient differentiation, insufficient alkalinity, etc). However, > everything I have been able to read on the topic suggests that > over-staining is "easily" differentiated from true amyloid staining by > using polarizing light microscopy. That is, true amyloid produces apple > green fluorescence while non-amyloid components produce silver/grey color. > > *My question:* > I want to know if anyone has encountered false positive staining that *is > apple green* in color? We had a few bone marrow core biopsies that stained > bright green but were later found to be negative when stained at another > lab. We subsequently threw out all of our working solutions and made up > everything fresh and repeated the previous (false positive) specimens and > they were indeed negative in our lab as well. > > *In order to prevent this from happening again, I need to attempt to > understand what may have caused this to happen in the first place. * > > This is where the vast collective knowledge of this group comes in. :-) > Can anyone offer some insight as to possible causes? > > *Our Congo Red method:* > > > Deparaffinize sections and bring them to water. > > Stain in Hematoxylin for 1 minute > > Add 0.5ml of 1% Sodium Hydroxide to 50 ml of stock alkaline salt solution. > > Wash slides in running water > > Place in *working* alkaline salt solution from step 2 for 20 minutes > > Add 0.5 ml of 1% Sodium Hydroxide to stock Congo red solution. > > Start to filter *working* Congo red solution when 15 mins are left in step > 6 > > Place sections in the *working* Congo red from step #8 for 20 minutes. > > Dehydrate the slides one at a time in 3 changes of absolute ethanol, 6 dips > each. > > Dip the slide 10 times in a coplin of xylene. > > Continue dehydrating the other slides. > > Coverslip the slides. > > *Greg Dobbin* > 1205 Pleasant Grove Rd > Route 220 > York, PE C0A 1P0 > _______________________________________________ > 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