[Histonet] Alzheimer stain

[Hofecker, Jennifer L]

Good Morning, Behnaz.
I am curious which modification of Bielschowsky you are using. Most of
the common modifications do not require Sodium Carbonate.
See below for my protocol which is based on Hirano's modified
Bielschowsky. Please let me know if you have any additional questions.
Good Luck! jennifer 

Jennifer L. Hofecker, HT(ASCP)
Vanderbilt University Medical Center
Division of Neuropathology 
Nashville, TN
ph. (615)343-0083
fax. (615)343-7089
NSH Quality Control Committee Chair
 
 
MODIFIED BIELSCHOWSKY METHOD

SOLUTIONS/REAGENTS
1.	20% SILVER NITRATE
	Silver nitrate............20 g
	Distilled Water.........100 ml
	Mix well and protect from light.  Prepare fresh. Fill one coplin
jar and set aside.

2.	AMMONIUM HYDROXIDE (CONCENTRATED)
	Commercially prepared
	
3.	REDUCED SILVER SOLUTION
      Add ammonium hydroxide, drop by drop, with constant stirring to
the remaining 20%
silver nitrate solution.  The solution will turn from dark brown to
black and then  	to clear. Filter through #1 filter paper ALWAYS
Prepare fresh.
	
4.	AMMONIA WATER
	Ammonium hydroxide.........2 drops
	Distilled water......62.5ml
	Prepare fresh.
	
5.	DEVELOPER (PREPARE IN THIS ORDER)
	Citric Acid.........0.25 g
	Distilled water......50ml
	Formaldehyde, 37%......10 ml
	Nitric acid...............25 uL
      Mix well after adding each item.  Stable for 5 days @ room temp.

6.	5% SODIUM THIOSULFATE
	Sodium Thiosulfate...............5 g
	Distilled Water..................100 ml


	
STAINING PROCEDURE

1.	Deparaffinize sections and hydrate to distilled water.
2.	Place slides 20% silver nitrate at room temp. for 30 min.
Protect from light. 
3.	Rinse at least 6 times in millipore double distilled water. Save
solution from step 
4.	Wipe moisture from slides and incubate in Reduced Silver
solution. Protect from 	light.
5.	Place slides directly in ammonia water.  Save reduced silver.
(Slides can remain in 	ammonia water for several minutes without
harming the staining.  Only 1-2 min are 	needed for the solution
to have desired effects.)
6.	Put 3 drops of developer in 30 ml of reduced silver (do not
prepare earlier).
7.	Place slides in this solution on a shaker near a microscope.
8.	Watch for sections to turn brown. Rinse slides in water.  Wipe
back of slide and 	check under microscope.  Plaques and tangles
should be black.  Check each slide 	individually.  If not dark
enough, return to ammonia water for 1 minute and back in 	reduced
silver/developer solution.  If silver starts to break down and form a
"mirror" on the coplin jar,  put 3 drops of developer in another 30 ml
of reduced 	silver (can use  solution from Step 4).  
9.	Wash in running water for 15 minutes.  Rinse in distilled water.
10.	Place in 5% sodium thiosulfate for 2 minutes.
11.	Wash in running tap water for 1 minute. Rinse in distilled
water.
12.	Dehydrate through graded alcohols.
13.	Clear in two changes of xylene, 3 min each.
14.	Mount with synthetic resin.


RESULTS
Senile Plaques and neurofibrillary tangles of Alzheimer's disease -
black
Neurons and background - brown to yellow.
Lipofuscin - brown or black

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Behnaz
Sohrab
Sent: Tuesday, April 01, 2008 1:55 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Alzheimer stain


Question regarding Sodium Carbonate Solution in Bielschowsky method for
Alzheimer. Can I substitute Carbonate? Or any one has a better method
for this stain? ( I do not have Sodium Carbonate, and no time to order
it). Thanks,Behnaz



_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet