Talk:Kubke Lab:Protocols/Hematoxylin Eosin

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Reuben:

re 'Modified' H&E procedure.

We didn't exactly modify the procedure. We changed the times spent in each solution to match our tissue but the procedure defines the solutions used and the order they were used. Everyone uses a 'modified' version of the procedure.

Satya asked to refer to it as modified --MF Kubke 21:25, 13 December 2010 (EST)

Contents

To decide what to move to the main page

Haematoxylin and Eosin staining procedure

This is the protocol established by Malisha Hettiarachchi and Reuben Cutfield

The tissues stained were chick embryos fixed in paraformaldehyde between stages 20-30 (Hamburger and Hamilton staging). The haematoxylin used was Gills 2. The tissues were often cryoprotected in 30% sucrose prior to sectioning and mounting.

1 dip each
Approximately 10 dips
# Haematoxylin Gils 2

30sec – 1min

  1. Tap water

Approximately 10 dips until all excess stain is removed

  1. Differentiate in 1% acid alcohol

1 Dips

  1. Tap water

Approximately 10 dips

  1. Blue in 1% Lithium carbonate

6 dips

  1. Tap water

Approximately 10 dips

  1. Stain in 1% Eosin

5-30seconds

  1. Absolute alcohol 1 and 2

Rapid dips

  1. Xylol 1,2 and 3

10 dips each

  1. Mount coverslip with DPX mount



1% Acid Alcohol

Preparation :

3.5L Absolute alcohol

850ml

1.5L distilled water

350ml

25ml HCL

6.5ML

Mix together and store in a cool place


Gils 2 Haematoxylin

Distilled water 730ml

Ethylene Glycol 250ml

HX 4gms

Sodium Iodate 0.4gms

Aluminium sulphate 70.4gms

Glacial acetic Acid 20ml

--------------------------------------------------------

1% Eosin

EosinY- 20gms

Distilled water- 2L

1% Calcium Chloride 20ml

----------------------------------------------------------

Note: Dissolve HX and Alum separately in distilled water and then mix.


1% Lithium Carbonate

Lithium carbonate 1gm

Distilled water 100ml

What goes into the main page

I think that the main page should have fail proof protocols, that we know work to satisfaction and the references to the material from where the information was obtained. In the meantime, discussions as to what should/should not gone it are better suited for the talk page --MF Kubke 21:25, 13 December 2010 (EST)


Moved from protocol page to talk page for clarification

Reuben where is this information from? Satya did not provide it on the protcol we used so I don't think it should be included. Was this the way Satya made the solutions for us ? --Malisha Hettiarachchi 22:24, 13 December 2010 (EST).

yes, this is how she made up the solutions. she emailed me the list.

Thanks for letting me know I will restore the old version --Malisha Hettiarachchi 22:38, 13 December 2010 (EST).

Gils 2 Haematoxylin

  • Distilled water 730ml
  • Ethylene Glycol 250ml
  • Pure Haematoxylin 4gms
  • Sodium Iodate 0.4gms
  • Aluminium sulphate 70.4gms
  • Glacial acetic Acid 20ml

1% Eosin

  • EosinY- 20gms
  • Distilled water- 2L
  • 1% Calcium Chloride 20ml

Dissolve Haematoxylin and Alum separately in distilled water and then mix.

1% Lithium Carbonate

  • Lithium carbonate 1gm
  • Distilled water 100ml

=Physiology=

Intro In general staining techniques act to utilize the inherent differences in the wavelength of absorbed light within a tissue to visualize its structure (Disbrey & Rack,1970). A dye absorbs light at particular wavelengths, the chromophore describes the coloured part and the auxochrome describes the point of attachment to the tissue (Disbrey & Rack,1970). Many auxochromes exist because of the acidic or basic properties of the dye (Disbrey & Rack,1970). A mordant is a chemical with a strong affinity for the dye and the tissue, this is most commonly a metal compound (Disbrey & Rack,1970).

This text would be more appropriate for a general description of histological techniques. Not sure how it is relevant to the protocol itself, since this information ins not specific to H&E. I also do not see why this would be considered 'physiology'.--MF Kubke 19:35, 13 January 2011 (EST)
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