[Histonet] Testes - Fixation, Staining, Staging

[bettina.hutz <@t> orionpharma.com]

 Hello,

Here instructions for HE and PAS after Hotchkiss my Pathologists likes;) We
fix testis in Boiun for 24 hours, then transfer them to 70% ethanol in which
they can be kept as long as you wish. We do not rinse them several times in
70% ethanol. If you wish I can ask him more about satging when he returns
from Christmas vacation.

Hope this helpes you:)

I can send you also pictures of my stainings if you like to see the result.

Bettina Hutz
Research Assistant
Department of Discovery Biology
Orion Corporation, ORION PHARMA
Orionintie 1, P.O.Box 65, FIN-02101 Espoo, Finland
Tel. +358 10 429 2607,  Fax +358 10 429 2924
Email: bettina.hutz <@t> orionpharma.com 



 

Department of Discovery Biology

Hemalaun-Eosin staining of paraffin slides

1.	Staining procedure
Deparaffination
3x xylene				each min. 3 minutes
2x ethanol 100%				short
ethanol 90%				short
ethanol 80%				short
ethanol 70%				short
aqua dest.				Short

Staining
Mayer´s Hemalaum			3 minutes
Running tap water			10 minutes
Rinse with aqua-dest.
Eosin					40 seconds

Dehydration & mounting
ethanol 70%				short
ethanol 90%				short
2x ethanol 100%			short
3x xylene	 			short
Mount with xylene based medium

2.	Solutions
2.1.	Mayer´s Hemalaunn

2.1.1	Stock solution 
Mayer´s Hemalaum (Fa. Merck)		

2.1.2.	Usage solution
Mayer´s Hemalaun			3 parts
Aqua-dest.				1 part
Filtrate before use

2.2  Eosin

2.2.1	Stock solution
Eosin Yellow					1,0 g
Aqua-dest.					1000 ml
 
2.2.2.	Usage solution
Eosin stock solution			50,0 ml
Aqua dest.				450 ml
+ 6 drops actetic acid 100%

3.	Results

Nuclei					- blue
background				- red

 

PAS reaction after Hotchkiss for paraffin slides

1.	Staining procedure

Deparaffination
3x xylene				each min. 3 minutes
2x ethanol 100%				short
ethanol 90%				short
ethanol 80%				short
ethanol 70%				short

Staining
Periodic acid	(oxidation)		5 minutes
70% ethanol				1 minute
Reduction solution			1 minute
70% ethanol				1 minute
Schiff´s reagent				20 minutes
Running tap water			10 minutes
Mayer´s hemalaun			3 minutes
Running tap water			10 minutes

Dehydration & mounting
ethanol 70%				short
ethanol 90%				short
2x ethanol 100%			short
3x xylene	 			short
Mount with xylene based medium

2.	Solutions

2.1.	Mayer´s Hemalaunn

2.1.1	Stock solution 
Mayer´s Hemalaum (Fa. Merck)		

2.1.2.	Usage solution
Mayer´s Hemalaun			3 parts
Aqua-dest.				1 part
Filtrate before use

2.2  Periodic acid

(produce short before use)
Periodic Acid				1,6 g
Aqua-dest.				40 ml
Ethanol 100% p.a.			140 ml
Sodiumacetate 0,2 N			20 ml

2.3  Reduction solution

Potassiumiodid				4 g
Sodiumthiosulfate			4 g
Aqua-dest.				80 ml
HCl,  2 N				2 ml

2.4  Schiff reagent

Schiff´s reagent (Fa. Sigma-Aldrich), 500 ml	
		
3.	Results

Nuclei					- blue
Carbohydrates				- pink-deep red