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.


CD4 foxp3 staining on mouse melanoma tumor tissue HIGH BACKGROUND PROBLEM

CD4 foxp3 staining on mouse melanoma tumor tissue HIGH BACKGROUND PROBLEM - Immunohistochemistry Forum

CD4 foxp3 staining on mouse melanoma tumor tissue HIGH BACKGROUND PROBLEM - 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 12-19-2011, 12:02 PM
Pipette Filler
Points: 62, Level: 1 Points: 62, Level: 1 Points: 62, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2011
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Smile CD4 foxp3 staining on mouse melanoma tumor tissue HIGH BACKGROUND PROBLEM



Hey,
I am a first year Phd student and I am trying to perform immunohistochemical staining on tumor tissue harvested from mouse injected with B16F10 melanoma cells.My protocol is as follows :
1. Harvest tissue and embed in OCT medium and freeze in -80C
2. Section of 10 u was taken on + coated slide
3. Section fixed in -20 cooled acetone for 5 min at RT
4. wash 3 times in PBS (5 min each)
5. Blocking with 10% horse serum at RT for 1h
6. Washed 2 times with PBS (5 min each)
7. Add primary Ab (Abcam Rabbit polyclonal to FoxP3 and Biotin Rat antimouse CD4 - both I have tried with 1:300 and 1:500 dilution) for 30 min at RT Ab diluent is PBS + 1%BSA
8. 4 wash with PBS (10 min each)
9. Add secondary Ab (Streptavidin -FITC and DyLight 549 Donkey anti rabbit IgG- I have tried dilution of 1:3000 and 1:8000) Ab diluent is PBS + 1%BSA Ffor 30 min at RT.
10 4 wash with PBS (10 min each)
11 Add DAPI (1:4000) FOR 5 MIN at RT in dark
12 wash with PBS FOR 5 MIN AT rt
13 Incubate with 1%PFA in PBS for 5 min
14 mount in mounting media

The problem is that there is there is high bacground and nonspecific staining . Almost everything seem to stain and even where there is no nuclear stain there is still red (foxp3)and green (cd4) staining I am absolutely confused what to do ... please help me with suggestion
Thanks in advance
Reply With Quote
Reply

Tags
background , cd4 , foxp3 , high , melanoma , mouse , problem , staining , tissue , tumor


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
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
>>>> LOWER CHOLESTEROL <<<< Janis Stroud Forum Physik 0 09-22-2009 04:39 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM


All times are GMT. The time now is 12:19 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 0.15523 seconds with 16 queries