Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Histology Forum > Immunohistochemistry Forum
Register Search Today's Posts Mark Forums Read

Immunohistochemistry Forum Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.


Entangling of freefloating tissues during staining

Entangling of freefloating tissues during staining - Immunohistochemistry Forum

Entangling of freefloating tissues during staining - Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-17-2011, 05:19 PM
Pipette Filler
Points: 255, Level: 5 Points: 255, Level: 5 Points: 255, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Posts: 8
Thanks: 0
Thanked 0 Times in 0 Posts
Angry Entangling of freefloating tissues during staining



Dear friends
I am new to this field and i am working from this feb on this topic

I will perfuse the mice brain(7day old pups) ,
take out the brain, fix it 24h in PFA
transfer to 1XPBS leave it for overnight at 4
embedd them in 3% agarose and take 40 micron sections
i wash them in wash buffer containing 0.1%triton-x100 in 1xPBS (10min)
block with 3%BSA, 10%NGS, 0.1%Triton-X100 in PBS (1h)
Primary antibody and secondary antobody-same blocking buffer except NGS

My tissues are very fragile and by the time i put them in the secondary they will become entangled, folded and unable to mount, i tryed to fig out the problem but disappointed plz can anyone help me in this regard

Madhu
Reply With Quote
  #2  
Old 03-11-2013, 10:34 PM
haisond's Avatar
Pipette Filler
Points: 80, Level: 1 Points: 80, Level: 1 Points: 80, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2013
Location: los angeles, CA
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Entangling of freefloating tissues during staining

Hi Skumar..

You are in luck, I've worked out all the issues with freefloating section IHC. I routinely stain 40-60 microns brain slices so i have become somewhat of an expert at this..

here are some solutions to your problem:
1. Buy a used Ronco rotiserie oven from Ebay (30 to 40 bucks), you can disable the heating element quite easily.
2. Buy a used well plate sealer ( I got one (Packard microplate sealer) for $50 bucks from Ebay).
3. Buy some thermal plate sealer film.
4. Place your specimens in 24-well plate, incubate in 750 ul - 1,000 ul of antibody solution
and seal wells with using the film and plate sealer. Your samples are now sealed and can be rotated in a rotiserie manner allowing for complete penetration of your tissue specimen from all sides, the constant motion will prevent tissue folding during incubation.
5. I place the rotisserie in a fridge to incubate samples at 4 degrees.
6. For mounting you should be concern with compression of your specimen by the weight of the coverslip. Buy some laminating film from McMaster Carr (catalog number 2271k72) these are 50.6 micron thick with an adhesive backing to tape onto your slide. Once the film is taped onto your slide, cut out an area large enough to fit your specimen. Basically, you will end up with a well that is 50.6 micron thick to place your specimen into. Place specimen into the well, use a one bristle brush (Electron Microscopy Science) to gently straighten out any wrinkling or folding. Once the specimen is in place remove excess liquid by dabbing with a kimwipe, do not let your sample dry out completely. Quickly add your mounting solution and coverslip with a 0 to 1 coverslip (EMS). I have many more tricks, if anyone else have issues with working with brain tissues.

good luck,
Reply With Quote
Reply

Tags
entangling , freefloating , staining , tissues


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Staining Of Tapeworms Razielox Basic Lab Protocols and Techniques 2 03-18-2011 03:02 PM
Gel Staining Tray Plastic Electrophoresis DNA Staining admin Lab Equipment Discussion and Reviews 0 03-08-2011 10:30 AM
Immidiate dissappearing of bands on DNA sequencing gels after silver staining himanshu SDS-PAGE Gel Electrophoresis Forum 2 07-24-2008 06:17 PM
whole mount antibody staining of muscle mena22787@excite.com Zebrafish Forum 0 03-05-2008 04:36 PM
glycoprotein staining chetan patel Protein Forum 0 08-22-2005 07:19 AM


All times are GMT. The time now is 10:14 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11929 seconds with 16 queries