[Histonet] IHC hand staining

[Hughes, Anna [JRDUS]]

Hi!
I have done a lot of manual staining in the past....a simple protocol is below:  Any of the Dako kits are wonderful...as are the Biocare

1. Depar slides as usual 
2.Place slides in DPBS (no mag or calcium) 2x 5 minutes
3. Antigen retrieval if necessary - will have to look at the insert for your primary antibody.
4. Block Peroxidases for 10 minutes with 3% H2O2
5. Wash using DPBS 2x 5minutes
6. Protein block - 30 minutes using 5% normal serum of the host of your secondary antibody
7.Wash using DPBS 2x 5 minutes
8. Place slides in your primary antibody (I usually use 200-300ul/slide depending on tissue size and cover with a small piece of parafilm).  Incubate in a humidity box at RT on the bench for 1 hour
9. Wash using DPBS 2x5 minutes
10. Place slides in a biotinylated secondary antibody (incubate same as primary above)- for 20 minutes
11. Wash using DPBS 2x5 mintues
12. Use a DAB detection kit such as the DAKO Envision kit - follow their directions in the kit. (These come as mouse,rabbit, rat...... so if you buy the kit as a whole, it comes with the biotinylated secondary (goat host) - already diluted and ready to use)
13. Wash 2x dH2O
14. Counterstain with Hematoxylin for 5 minutes
15. Wash with dH2O 2x 5 minutes
16. Blue slides  -this will depend on your hematoxylin
17. Wash with dH2O 2x5minutes
18. Dehydrate through alcohols to xylene and coverslip.

Hope this is helpful!
Anna Hughes


-----Original Message-----
From: Jennifer MacDonald [mailto:JMacDonald at mtsac.edu] 
Sent: Thursday, November 05, 2015 6:20 PM
To: Elaine allison Hoffman
Cc: Histonet List
Subject: Re: [Histonet] IHC hand staining

In one of our classes the students do IHC staining by hand.  They perform multiple procedures over the semester.  We currently use Biocare reagents. 
 The polymer based detection systems are simple to use.  The data sheets that accompany the antibodies and detection reagents are really helpful with protocols. 



From:   Elaine allison Hoffman via Histonet 
<histonet at lists.utsouthwestern.edu>
To:     Histonet List <histonet at lists.utsouthwestern.edu>
Date:   11/05/2015 08:08 AM
Subject:        [Histonet] IHC hand staining



Greetings Histonetters,
I need some information on "hand" immuno-staining.  We are a small GI lab doing Giemsa stains on H. Pylori right now, but the Lab Director wants me to look into IHC hand staining for H.Pylori.  Anybody out there doing hand staining and willing to give me a protocol to try? Or maybe point me in the right direction to who could help me with that.  Any reliable IHC staining kits recommended?  Thanks in advance for the help....
Elaine Hoffman
The Gastroenterology Clinic & Endoscopy Center, IncGI Pathology 
Laboratory1622 E Market StreetWarren, Ohio   44483

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