[Histonet] Immunofluorescent staining of IgG on kidney frozen sections---High background!

[yichao wu]

Dear Dr.Callis,Dr.Yaskovich,,Dr. Li,and all histonetters,

Thank you very much for all of your reply.The frozen sections we make now 
have better mophology with Liquid N2 freezing.

Still three questions with regarding to kidney immunofluorescent staining.

1) We stain human IgG on kidney frozen sections with antibody from DAKO 
(F0202),and dilution is 1:50,but the background is always strong.

Other antibodies to IgA(F0204 from DAKO),IgM(F0203 DAKO),C3(F0201 
DAKO),C4(F0169 DAKO)&C1q (F0254) all produce dark background under 
fluorescent microscope,which is highly preferred.

Why IgG is so strange? Any problem with the antibody? Or the unspecific 
staining for IgG is special?

2) What kind of O.C.T is the best?

Thank you very much!

Yichao WU
Research Institute of Nephrology
Jinling Hospital
Nanjing 210002
P.R.China



>From: Terry Li <tli1 <@t> flowcity.bsd.uchicago.edu>
>To: "yichao wu" <yichaowu <@t> hotmail.com>
>Subject: Re: Please help with me--routine immunofluorescent staining on  
>kidney specimens
>Date: Thu, 18 Mar 2004 11:35:36 -0600
>
>Yichao,
>
>Autofluoresence is a big disadvantage from paraffin tissues.  Chosing 
>fixatives is also important depend on what protein you are dealing with.
>
>My answer for your first question: It's very easy to get the block out of 
>the plastic mold.  Use your finger to warm the bottom of the block, then 
>you can get it out easily.  Add OCT on the tissue holder (chuck) and put 
>the tissue block on top of it.  They will stick together in cryostat and 
>your tissue is ready to be cut.
>
>Second question: Store the frozen samples in -80oC freezer until to use.  I 
>am working on a hand book about laboratory methods with English versus 
>Chinese.  Here is part of it.  I hope it may be helpful for you.  "Feed 
>back" is very welcome.
>
>Immunohistochemistry on Frozen Tissue
>The best method for antigen preservation in immunohistochemistry is 
>freezing.  Freezing is a very suitable method for preserving antigens which 
>lose their immunoreactivity.
>
>1. Preparation of Frozen Tissues for sectioning
>  Materials:
>           2-methylbutane;
>           Dry ice;
>           Plastic molders;
>           Frozen tissue matrix (O.C.T. compound, 4583 from SAKURA);
>           Long forceps;
>           Metal or plastic tall cylinder.
>
>Methods:
>1)    Pour 2-methylbutane into a metal or plastic tall cylinder.  Keep 
>adding small pieces of dry ice into the solution to make the temperature 
>cool down to -40-50o C and keep it for certain long time as desired.
>2)    Label plastic models and fill the bottle with O.C.T..
>3)    Collect desired tissues.  Preserve the best tissue integrity is the 
>key to have high quality of immunohistochemistry result.  Have fine tools 
>and use fine technique.
>4)    Place tissue into the labeled plastic model and fill the molder with 
>more O.C.T. until the tissue get fully covered.  Arrange tissue in the 
>matrix near the bottom so tissue is easily exposed when sections are cut, 
>and adjust the orientation if desire.  Place molder with tissue into the 
>cylinder of cold 2-methylbutane.  Allow the tissue matrix to solidify from 
>the bottle of the molder to the top to avoid bubbles and leave it in the 
>solution for 5-10 minutes.
>5)    Store blocks in the -80oC freezer until ready sectioning.
>
>Note:
>1.     Get rid of extra aqueous from tissue specimen with paper towel, do 
>not wash tissues before frozen.
>2.     Some tissues need to be fixed first.  Tissue samples can be treated 
>with 10%-30% sucrose-PBS after fixation, and then frozen as above.
>
>2. Sectioning of Frozen Tissues
>
>Materials:
>Frozen tissue block;
>Superfrost plus slides (12-550-15 from Fisher Scientific);
>Blade (low or high-profile disposable blades or non-disposable blade);
>Frozen Tissue Matrix (O.C.T.).
>
>Instrumentation:
>Cryostat (Leica, Microm or other brands).
>
>Methods:
>1)     Before cutting sections, allow the temperature of the frozen tissue 
>block to equilibrate to the temperature of the cryostat (-20oC in general).
>2)     Use O.C.T. to freeze tissue block on the specimen disk and fix the 
>disk on the machine.  Adjust the positioning of the block to align the 
>block with the knife blade.  Cut the tissue until the desired tissue is 
>exposed and start collection.  Put sections on super plus slides and air 
>dry over night.  The thickness of sections is various depend upon your 
>research.  3-6 um sections are commonly used.
>3)     Fix the sections by immersion in cold acetone/methanol for 20 min, 
>air dry and start the staining procedure or store the slides at sealed 
>slide box in -80oC until you are ready.  Or store the slides without any 
>fixation.
>4)    Use O.C.T. to cover exposed tissue and store it in -80oC.
>
>
>Shihong Li
>Immunohistochemistry Faciltiy
>University of Chicago
>
>
>
>
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