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Author Topic: immunofluorescence staining  (Read 7003 times)

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Offline fery

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immunofluorescence staining
« on: September 14, 2005, 06:10:49 AM »
Hi All

I want to stain a paraffin embedded tissue slide with antibody conjugated with FITC and PE ( they are specific for my cell but they are for flow cytometry use). I appreciate for any help, comment and protocol

Regards


Fery

immunofluorescence staining
« on: September 14, 2005, 06:10:49 AM »

Offline richard03

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immunofluorescence staining
« Reply #1 on: September 14, 2005, 07:19:26 AM »
What antibodies? Manufacturers?

Offline fery

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NKT antibody
« Reply #2 on: September 14, 2005, 09:19:42 AM »
Dear Richard


My antibodies are from BD biosciense pharmingen and here is datasheet for one of them, the other is anti CD3 gojugate with different dye(PE)

PRODUCT INFORMATION
Catalog Number: 552824
Size: 0.1 mg
Clone: 6B11
Antibody Composition: Mouse IgG1, κ
WS No.: NA
Storage Buffer: Aqueous buffered solution containing 0.09% sodium azide.
DESCRIPTION
Monoclonal antibody 6B11 reacts with the CDR3 region of the invariant TCRα chain (Vα24-JαQ), expressed on invariant NK T
cells. Invariant NK T cells are a small subset of lymphocytes that also express other NK cell molecules such as CD161 and
have been reported to play an immunoregulatory role. The Vα24-JαQ interacts with the CD1d protein. Invariant NK T cells
can be specifically activated by CD1d-presented antigen resulting in rapid production of IL-4 and IFN-γ. Studies of NK T cells
describe their role in anti-tumor immunity and autoimmunity.1-3
PREPARATION AND STORAGE
The antibody is purified from tissue culture supernatant by Protein G affinity
chromatography.
Store at 4C.
USAGE
This reagent is effective for indirect immunofluorescence staining of human
tissue for flow cytometric analysis. Since applications vary, however, each
investigator should titrate the reagent to obtain optimal results. Note: this
product is routinely tested on peripheral blood lymphocytes (CD3+). The
histogram is an example of the expected reactivity.
Recommended Isotype Control: Mouse IgG1, κ, clone MOPC-21,
Cat. No. 555746
Recommended Second Step Antibody: Goat-anti-Mouse IgG/IgM-FITC,
Cat. No. 555988
CD3-APC
101 102 103 104 100

Offline richard03

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immunofluorescence staining
« Reply #3 on: September 14, 2005, 09:32:32 AM »
It looks to me that this antibody is not Fluorescence-conjugated. You will still need a second layer fluorescence-conjugated antibody. The following protocol for your reference:

http://www.ihcworld.com/_protocols/general_IHC/immunofl.htm

You may need to use antigen retrieval method for pretreatment.

Richard

Offline fery

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FITC Conjugated Mouse Anti-human Invariant NK T Cell
« Reply #4 on: September 14, 2005, 12:41:18 PM »
Dear Richard

Excuse me for my mistake about datasheet. here is datasheet of FITC Conjugated Mouse Anti-human Invariant NK T Cell:

Product Information
Catalog Number: 558371
Size: 100 tests
Clone: 6B11
Isotype: Mouse IgG1, κ
WS No. NA
Storage Buffer: Aqueous buffered solution containing BSA* and 0.09% sodium azide.
*Source of all serum is from the United States.
Description
Monoclonal antibody 6B11 reacts with the CDR3 region of the invariant TCRα chain
(Vα24-JαQ), expressed on invariant NK T cells. Invariant NK T cells are a small subset
of lymphocytes that also express other NK cell molecules such as CD161 and have been
reported to play an immunoregulatory role. The Vα24-JαQ interacts with the CD1d
protein. Invariant NK T cells can be specifically activated by CD1d-presented antigen
resulting in rapid production of IL-4 and IFN-γ. Studies of NK T cells describe their role
in anti-tumor immunity and autoimmunity.1-3
Preparation and Storage
The antibody is purified from tissue culture supernatant by affinity chromatography and
is conjugated with FITC under optimum conditions. The solution is free of unconjugated
FITC.
Store conjugate at 4 C and avoid prolonged exposure to light. Do not freeze.

Usage
This reagent is effective for direct immunofluorescence staining of human tissue for flow cytometric analysis using 20 l/106 cells. Since applications vary, each investigatorshould titrate the reagent to obtain optimal results. Note: this product is routinely testedon peripheral blood lymphocytes (CD3+).
Recommended Isotype Control: Mouse IgG1, κ, clone MOPC-21, Cat. No. 555748.


do you think that with this antibody I could do my job

Regards

Fery

Offline richard03

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immunofluorescence staining
« Reply #5 on: September 14, 2005, 01:05:31 PM »
Hi Fery,

The datasheet does not mention anything about paraffin section neither immunohistochemistry. I would suggest to start with Tris-EDTA pH9 antigen retrieval pretreatment and high concentration of primary antibody (such as 1:25-1:50).

Richard

Offline fery

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secondary antibody
« Reply #6 on: September 14, 2005, 04:33:25 PM »
Dear Richard

Thank you very much for your advice but please let me know if I need any second step antibody or isotype control

Regards

Offline richard03

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immunofluorescence staining
« Reply #7 on: September 14, 2005, 05:17:16 PM »
Fery,

You don't need second layer antibody if you used FITC-conjugated primary ab. You can use same isotype as your primary for negative control.

Richard

Offline ImmunoNYC

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immunofluorescence staining
« Reply #8 on: September 15, 2005, 01:32:16 AM »
You don't *need* a second layer but you may want to use one if your signal is very dim. In such cases, I use anti-FITC secondary available from numerous different sources conjugated to just about anything including Alexa 488 (another green fluorophore like FITC). It will give amplification of a potentially dim signal!

Offline richard03

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immunofluorescence staining
« Reply #9 on: September 15, 2005, 09:07:33 AM »
Good point! MaximinaNYC. Thanks.

Richard

immunofluorescence staining
« Reply #9 on: September 15, 2005, 09:07:33 AM »