What concentration, how long and at what temperature do you 
recommend?  Sorry for the ignorance but I have never tried pepsin 
for ISH before.
Thanks.
Agripina
 
From:           	Patsy.Ruegg@UCHSC.edu
To:             	ASuarez@mrl.ubc.ca
Subject:        	RE: ISH of VEGFs and their receptors
Date sent:      	Thu, 3 Feb 2000 12:32:00 -0700 

> Proteinase K can be difficult to work with, try pepsin instead.
> Patsy Ruegg
> 
> -----Original Message-----
> From: Agripina Suarez [mailto:ASuarez@mrl.ubc.ca]
> Sent: Wednesday, February 02, 2000 4:23 PM
> To: Histonet@pathology.swmed.edu
> Subject: ISH of VEGFs and their receptors
> 
> 
> 
> Histonetters, help!!!
> I have been trying to do in situ hybridization of VEGFs  (A, B, C, D) 
> and their receptors (Flt-1, -4 and Flk-1) in formalin-fixed, paraffin-
> embedded normal human vascular tissues and transplant heart 
> vessels but still unsuccessful so far. I have tried optimization of the 
> proteinase K concentration (2, 5, 10 and 20 ug/mL) and its 
> incubation times (15, 30, 45, 60 min) at 37oC. I have also tried 
> prolonged incubation in color substrate (BCIP/NBT, 1 Sigma 
> tablet/200 mL dH2O) at 37oC up to 5 days. The riboprobes were 
> labeled with digoxigenin and prepared by in vitro transcription. The 
> efficiency of transcription and labeling were confirmed by gel 
> elctrophoresis and  dot blot hybridization. I use 3-4 ug/mL of 
> riboprobes, 20 uL applied on tissue.  I use only DEPC water in my 
> solutions and all glassware have always been oven-treated for at 
> least 3 hours at 200oC. My positive tissue controls which are 
> kidney carcinoma, fetal kidney, endometrium and term placenta 
> were always negative while my run controls (CVB3 riboprobes on 
> CVB3-infected mouse tissues) were always positive. The protocol I 
> use is as follows:
> 
> -Dewaxing in 3 x 5 min in xylene
> -Rehydration in ethanol series  (90, 70, 40%, DEPC water)
> -0.2N HCl for 20 min, brief rinse in DEPC water
> -2x SSC, 30 min, RT
> -Proteinase K (tried 2, 5, 10, 20 ug/mL in .2M Tris pH 7.2, 50 mM 
> EDTA pH 8), 37oC for 15, 30, 45, or 60 min
> -100 mM triethanolamine pH 8 with 0.25% acetic anhydride, 2 x 5 
> min
> -2x SSC, 5 min
> -Dehydration in series of ethanol, air dry slides
> -Hybridization in humid chamber, 18 hrs, 42oC ( will be trying 
> longer incubation times)
> -50% formamide in 2x SSC, 50oC, 30 min
> -2x SSC, 50oC, 2 x 20 min
> - 0.2X SSC, 50oC, 2 x 20 min
> - 100 mM Tris pH 7.5 w/ 150 mM NaCl (buffer 1), 10 min
> - 2% lamb serum in Buffer 1, 30 min
> - Incubation in Anti-dig-AP, 30 min, 37oC
> - Buffer 1, 3 x 5 min
> - 100 mM Tris w/ 100 mM NaCl and 50 mM MgCl2, pH 9.5, 10 min
> - Incubate in color substrate (BCIP/NBT, 1 tablet/200 ml dH2O ) 
> overnight or up to 5 days
> - Wash with dH2O, dehydrate, counterstain.
> 
> Any advice, pointers you can give me to achieve positive results for 
> the VEGFs would be greatly appreciated.
> 
> Agripina
> 
> 
> 
>  
> 
> 
> 
> 
> 
> Agripina C. Suarez
> Cardiovascular Research Laboratory
> Room 292, McDonald Research Wing, St. Paul's Hospital
> 1081 Burrard St., Vancouver
> B.C., Canada V6Z 1Y6
> Phone: (604) 806-8659
> Fax:   (604) 806-8351 


Agripina C. Suarez
Cardiovascular Research Laboratory
Room 292, McDonald Research Wing, St. Paul's Hospital
1081 Burrard St., Vancouver
B.C., Canada V6Z 1Y6
Phone: (604) 806-8659
Fax:   (604) 806-8351