Greetings Sujata:
On 3/31/2011 1:39 PM, Sujata Sovani wrote:
Hi All,
I am new to histology.
I came across the protocol below for staining GAGs (I plan to use that for
alginate) in an online book 'Theory and practice of histological techniques',
By John D. Bancroft, Marilyn Gamble.
A good book.
The protocol is by Hollander dated 1963.
A very old, but not necessarily bad, method. Why do you want to use this
particular method? There are simpler ways to do this.
Alcohol fixation might retain GAGs better ... how about staining with 1%
Alcian Blue in 3% acetic acid?
Would like to know if anyone has used this protocol before? Any tips/specific
details might be helpful.
The 20% HCl - does it mean 20% of 37.3% concentrated hydrochloric acid?
Yes.
Any details about acriflavine to be used would be helpful.
Can't help you there.
I'll bet DMAB is very toxic.
Geoff
Thank you.
Regards,
Sujata
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The protocol is as below:
Protocol
Acriflavine-DMAB method for sulfatide (Hollander 1963)
Fixation and sections
Post-fixed cryostat sections; formal calcium-fixed frozen sections
Preparation of reagents
1.Acriflavine stock solution
Acriflavine 100mg
Distilled water at 80 deg C 20ml
Store in dark at 4 deg C
2.Acriflavine working solution
0.1M citrate-HCl buffer pH 2.5 99ml
Stock acriflavine solution 1ml
3.DMAB solution
p-dimethylaminobenzaldehyde 0.6g
20% HCl 30ml
Isopropanol 70ml
Method
1. Mount sections onto slides
2. Stain for 6 minutes in acriflavine solution
3. Differentiate for 1 minute in two changes of 70% isopropanol
4. Treat with DMAB reagent for 30-45 seconds
5. Rinse in distilled water for 2-3 minutes
6. Counterstain nuclei in Mayer’s or Carazzi’s hematoxylin
7. Blue in tap water, rinse in distilled water and mount sections in
glycerin jelly.
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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mcaul...@umdnj.edu
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