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Loewe: Protocol For Immunofluorescence (IF) Assay

This document provides a protocol for performing an indirect immunofluorescence assay to visualize plant pathogenic bacteria. It describes sample preparation, formulations of buffers used in the assay, handling and storage of reagents, and the step-by-step assay procedure.

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Aditi Mohan
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98 views3 pages

Loewe: Protocol For Immunofluorescence (IF) Assay

This document provides a protocol for performing an indirect immunofluorescence assay to visualize plant pathogenic bacteria. It describes sample preparation, formulations of buffers used in the assay, handling and storage of reagents, and the step-by-step assay procedure.

Uploaded by

Aditi Mohan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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®

LOEWE

Protocol for Immunofluorescence (IF) Assay

LOEWE® Biochemica GmbH, Mühlweg 2a, D-82054 Sauerlach


Contact Germany Phone +49-(0)-81046 1620 E-mail: Service@loewe-info.com Web: www.loewe-info.com
Rev15.04.2015 1
Assay Principle
Immunofluorescence is a technique based on an antigen-antibody reaction where the
antibodies are tagged (labelled) with a fluorescent dye and the antigen-antibody complex
is visualized using ultra-violet (fluorescent) microscope. Therefore it allows the
visualization of the distribution of the target molecule through the sample. This protocol
describes how to perform an indirect immunofluorescence assay for plant pathogenic
bacteria.
1)
European and Mediterranean Plant Protection Organization (EPPO) PM 7/97 (1). Indirect immunofluorescence test for
plant pathogenic bacteria.

Sample preparation
According to international or laboratory standards.

Formulations of Buffers
.
Na2HPO4 12H2O 1.07 g
NaH2PO4.2H2O 0.4 g
IF-Buffer [10 mM phosphate buffered
NaCl 8.0 g
saline (PBS), pH 7.2]
KCl 0.2 g
For dilution of antibodies, antiserum and
Distilled water 1.0 l
positive controls and washing the slides.
Dissolve ingredients, check pH and sterilize by
autoclaving at 121°C for 15 min.
IF-Buffer-Tween
Add 0.1% Tween 20 to the IF-Buffer.
This buffer is used for washing the slides.
Phosphate buffered glycerol (Cover Na2HPO4.12H2O 3.2 g
solution), pH 7.6 NaH2PO4.2H2O 0.15 g
This buffer is used as a mountant fluid on the Glycerol 50 ml
windows of IF slides to enhance fluorescence. Distilled water 100 ml

Handling and Storage of Reagents

The following conditions are recommended for long-term storage:

 Antisera: prepare suitable aliquotes and freeze at min. -20°C.


 Secondary antibodies: Store lyophilizated antibodies refrigerated until opened. For
extended storage after rehydration, aliquot and freeze at min. -70°C. Alternatively,
add en equal volume of glycerol for a final concentration of 50% and store at -20°C
as a liquid. Note: adding glycerol reduces the stated protein concentration and
dilution range by one-half.
 Positive Controls: Store lyophilized controls refrigerated: Before use reconstitute as
indicated in the corresponding product specification sheet. Prepare aliquots and
store them frozen until use.

As a general rule, repeated thawing and freezing should be avoided!

LOEWE® Biochemica GmbH, Mühlweg 2a, D-82054 Sauerlach


Contact Germany Phone +49-(0)-81046 1620 E-mail: Service@loewe-info.com Web: www.loewe-info.com
Rev15.04.2015 2
Assay Procedure
Dilution of antiserum: according to the recommended working dilution*
Dilution of secondary antibody: according to the recommended working dilution*
Dilution of the positive control: according to the product specific data*

(* recommended working dilutions are stated in the corresponding product specification sheet)

Sample application
Pipette 20 µl of sample per window of the multiwell microscope slide and fix the samples
by heating at 60°C for 20 minutes using a thermo-stated heating plate.

Application of antiserum
Apply 20 µl of the diluted antiserum per window. Incubate the slides in a humid chamber
for 30 minutes at room temperature.

1st Washing step


Rinse the slides carefully with IF-Buffer-Tween. Wash the slides two times for 7 minutes
with IF-Buffer. Rinse with distilled water and carefully remove excess moisture (e.g. drying
in an incubator at 37°C).

Application of secondary labeled antibody


Apply 20 µl of the diluted secondary antibody per window. Incubate the slides in the dark
(to avoid bleaching) in a humid chamber for 30 minutes at room temperature.

2nd Washing step


Rinse the slides carefully with IF-Buffer-Tween. Wash the slides two times for 7 minutes
with IF-Buffer. Rinse with distilled water and carefully remove excess moisture (e.g. drying
in an incubator at 37°C).

Evaluation
Pipette 5-10 µl of the phosphate buffered glycerol per window or distribute a sufficient
amount across the slide, apply a coverslip and avoid exposure of the slides to excess
light. Examine the slides on an epifluorescence microscope with suitable filter
combination. Magnification: 400 – 1000 (immersion oil is necessary).

LOEWE® Biochemica GmbH, Mühlweg 2a, D-82054 Sauerlach


Contact Germany Phone +49-(0)-81046 1620 E-mail: Service@loewe-info.com Web: www.loewe-info.com
Rev15.04.2015 3

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