Talk:Kubke Lab:Protocols/Hematoxylin Eosin
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)
To decide what to move to the main page
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 |
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Approximately 10 dips until all excess stain is removed |
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1 Dips |
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Approximately 10 dips |
|
6 dips |
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Approximately 10 dips |
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5-30seconds |
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Rapid dips |
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10 dips each |
|
|
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)