AW: [Histonet] Histonet mailing list submission: Jones' Methenamine Silver Stain

[Gudrun Lang]

Hi Anna!
We had good success with following protocol:

Deparaffination, rehydration to dest. Water
0,5 % Periodic acid 20 min
Prewarm Methenamin-Silver-Solution without Borax in 60°C oven
Rinse slides in dest. Water 3x 5 min
Add Borax to Methenamin-Silver Solution
Put slides into Methenamin-Silver-Working solution for 45 min 
Prewarm coplin jar with dest. Water in 60°C oven
Rinse slides in prewarmed water
Let cool for 10 min
0,1 % goldchloride 10 min
Rinse in dest. Water 
2% Sodiumthiosulfate 5 sek
Rinse in running tapwater 5 min

Counterstain with hematoxylin if wanted.
Etc,

Methenamin-silver-Stock:
400 ml 3% hexamethylentetramin (CAS 100-97-0)
10 ml 10% silvernitrate (CAS 7761-88-8)
Solution turns first cloudy, then clear. Usefull for half a year.

Methenamin-silver-Workingsolution:
80 ml Silver-Stock
8 ml Borax (=sodiumtetraborate CAS 1303-96-4)
Gives a pH of 9

During incubation in silversolution a "mirror" developes onto the slides and
the coplin jars. Just when this point is reached, the reaction is enough and
ready.
This turning point occurs suddenly. You have to watch the jar every few
minutes after the first 40-45 min. 
Silversolution without Borax and pH 9 bring no silver-reaction.

Hope this helps
Gudrun


-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Anna
Huntley Coffey
Gesendet: Montag, 15. Juli 2013 23:13
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] Histonet mailing list submission: Jones' Methenamine
Silver Stain

Dear Histonetters,

I was wondering if anyone has experience with Jones' Methenamine Silver
stain?  We have attempted this stain several times, each time with a slight
tweak to the protocols we found online.  We found that it took much longer
than expected (3-4 hours compared to the expected 1 hour) for the membranes
to develop the expected black color every attempt we made.  The few times we
did see the black color develop in the membranes, the next step in the gold
chloride solution (1 minute) washed away all the dark color in the kidney,
leaving it a very light brown.  However, we did notice that only the skin
sample on our test slides retained the black color.  Since we ultimately
need this stain for kidney samples, this didn't do us any good.
 We tried thinner sections (2 micron), acid washed glassware, brand new
reagents, and multiple protocols with no luck.  I would greatly appreciate
any advice as to what we could try differently or if anyone else has
experienced these same problems.

Thank you!
Anna
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