Immunocytochemistry

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*Formaldehyde Fixation
*Formaldehyde Fixation
-
On the day of fixation, prepare a 4% solution of para-formaldehyde from a 16% stock solution[http://emsdiasum.com/microscopy/products/preparation/cover.aspx#15710, (Cat. #15710 also from Electron Microscopy Sciences)] in PBS (Gibco Cat. #14190-144).  It is important that the PBS and para-formaldehyde are sterile and mixed under the tissue culture hood.  This is the last step that must be performed with typcial tissue culture caution.
+
On the day of fixation, prepare a 4% solution of para-formaldehyde from a 16% stock solution[http://emsdiasum.com/microscopy/products/preparation/cover.aspx#15710, (Cat. #15710 also from Electron Microscopy Sciences)] in PBS (Gibco Cat. #14190-144).  It is important that the PBS and para-formaldehyde are sterile and mixed under the tissue culture hood.  This is the last step that must be performed with typical tissue culture caution.
Also, the para-formaldehyde solution forms a complex polymer structure which degrades very rapidly (within hours) and becomes "less" effective as a fixing reagent.  I like to freeze leftover 4% para-formaldehyde at -20, and have had success using frozen reagents up to 7 days later.
Also, the para-formaldehyde solution forms a complex polymer structure which degrades very rapidly (within hours) and becomes "less" effective as a fixing reagent.  I like to freeze leftover 4% para-formaldehyde at -20, and have had success using frozen reagents up to 7 days later.
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Fix for 15 minutes, no shaking.   
Fix for 15 minutes, no shaking.   
-
Rinse with 1X PBS (KD Medical Cat# RGF-3210: 10X) and then wash three times, five minutes each. I like to use an aspirator on my benchtop to suck off the liquid waste, and then a little squirt bottle with PBS to wash.  I try not to squirt directly onto the cover slips with the cells.  DO NOT LET THE CELLS DRY OUT.  Never leave the cells without liquid for more than 10 seconds.
+
Rinse with 1X PBS (KD Medical Cat# RGF-3210: 10X) and then wash three times, five minutes each. I like to use an aspirator on my bench-top to suck off the liquid waste, and then a little squirt bottle with PBS to wash.  I try not to squirt directly onto the cover slips with the cells.  DO NOT LET THE CELLS DRY OUT.  Never leave the cells without liquid for more than 10 seconds.
*Methanol Fixation
*Methanol Fixation
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==Block Cells==
==Block Cells==
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After removing the last wash, I add 10% Serum from the same mammal that the secondary antibody is produced from.  I typically use goat secondary antibodies, so I use 10% Goat Serum.  I purchase 100% goat serum, aliquot 10X10ml, and freeze all but one at -20.  Then, I typically leave only one aliqout at 4 degrees.  Leave overnight at 4 degrees.
+
After removing the last wash, I add 10% Serum from the same mammal that the secondary antibody is produced from.  I typically use goat secondary antibodies, so I use 10% Goat Serum.  I purchase 100% goat serum, aliquot 10X10ml, and freeze all but one at -20.  Then, I typically leave only one aliquot at 4 degrees.  Leave overnight at 4 degrees.
==Primary Antibody Incubation==
==Primary Antibody Incubation==

Revision as of 15:38, 14 June 2006

Contents

Introduction

Immunocytochemistry (ICC) is the process of taking tissue culture cells and applying antibodies to determine the spatial and temporal features of a particular antigen (often protein). A survey of the literature reveals many different techniques. However, there is often very little comment about the individual steps of the techniques, and hopefully this page will lead to a productive discussion. Often different techniques applied to the same antibody will reveal different results.

Immunofluorescence (IF) is a subset of ICC in which the antibody localization is visualized by fluorescence. The below protocol is focused on indirect IF (the use of a secondary antibody).

The Seven Basic Steps

  1. Grow Cells
  2. Fix Cells
  3. Permabalize Cells
  4. Block Cells
  5. Incubate with Primary Antibody
  6. Incubate with Secondary Antibody
  7. Place on a slide

Grow Cells

First, I place glass cover-slips (Cat. #63750-01 from Electron Microscopy Sciences) in the wells of a 6-well dish (#353046 from BD Labware). The key point to be made here is to not have too many or too few cells on the day you harvest the cells. Of course it will vary depending on the cell type, but for Hos and NIH-3T3 cells which I use, I like to plate .5 ml of a 10 ml confluent culture in a 6-well dish containing 1.5 ml media (total volume of 2 ml). I then let grow for 24 hours.

Fix Cells

  • Formaldehyde Fixation

On the day of fixation, prepare a 4% solution of para-formaldehyde from a 16% stock solution(Cat. #15710 also from Electron Microscopy Sciences) in PBS (Gibco Cat. #14190-144). It is important that the PBS and para-formaldehyde are sterile and mixed under the tissue culture hood. This is the last step that must be performed with typical tissue culture caution.

Also, the para-formaldehyde solution forms a complex polymer structure which degrades very rapidly (within hours) and becomes "less" effective as a fixing reagent. I like to freeze leftover 4% para-formaldehyde at -20, and have had success using frozen reagents up to 7 days later.

Fix for 15 minutes, no shaking.

Rinse with 1X PBS (KD Medical Cat# RGF-3210: 10X) and then wash three times, five minutes each. I like to use an aspirator on my bench-top to suck off the liquid waste, and then a little squirt bottle with PBS to wash. I try not to squirt directly onto the cover slips with the cells. DO NOT LET THE CELLS DRY OUT. Never leave the cells without liquid for more than 10 seconds.

  • Methanol Fixation

Block Cells

After removing the last wash, I add 10% Serum from the same mammal that the secondary antibody is produced from. I typically use goat secondary antibodies, so I use 10% Goat Serum. I purchase 100% goat serum, aliquot 10X10ml, and freeze all but one at -20. Then, I typically leave only one aliquot at 4 degrees. Leave overnight at 4 degrees.

Primary Antibody Incubation

Acknowledgments

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