[Histonet] Acriflavine staining of alginate for mucopolysaccharides

[Geoff McAuliffe]

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
>
> ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
>
> 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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mcauliff <@t> umdnj.edu
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