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Embryonic Stem Cell (ESC)/Induced Pluripotent Stem Cells (iPSC)

Anti SeV staining protocol

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Title Anti SeV staining protocol
Date Submitted April, 2012
Submitted by - Sunita DSouza
Adapted from -
Contributors - Vera Alexeeva, Sunita DSouza.
Affiliation(s) - Mount Sinai School of Medicine

Table of Contents

  1. Introduction
  2. FlowChart
  3. Materials
    1. Materials and Preparation
    2. Method- Live staining
    3. Method – Fixed staining
  4. Acknowledgements
  5. Contributors

Introduction:

Sendai virus (SeV), also known as murine parainfluenza virus type 1, is a negative sense, single-stranded RNA virus of the paramyxovirus subfamily Paramyxovirinae, genus Respirovirus, members of which primarily infect mammals. The SeV now has been developed as gene transfer vectors for expressing foreign genes to a wide range of mammalian cells and tissues with high efficiency.

Flowchart:

flowchart

REAGENT PREPARATION

Reagent Company Catalog#
1. 10XPBS Invitrogen 14200166
2. Saponin Sigma S4521-10G
3. HEPES 1M Sigma 15630-080
4. Sodium Azide Sigma S2002-25G
5. Glucose Sigma G7021
6. Triton X Sigma X100-100ML
7. PFA Electron Microscopy 15710
8. BSA 7.5% Invitrogen 15260-037
9. 1° Ab - Abcam chicken IgY Abcam Abcam 33988
10. goat anti chicken IgY-488 Abcam ab96951
11. rabbit SV antiserum MBL ab96951
12. donkey anti rabbit AlexaFluor 647 Invitrogen A21208

SAPONIN MEDIA

Company Working conc Working conc
PBS-10X Invitrogen 1X 50ml
Glucose Sigma 10gm/liter 5gm
BSA (7.5%) Invitrogen 0.2% 13ml
10%NaN3 Sigma 0.02% 1ml
1M HEPES Sigma 5mM 2.5ml
Saponin Sigma 0.5% 0.25gm
Water Invitrogen 433

SAPONIN MEDIA – 0.2% Triton X 100

Company Working conc Working conc
PBS-10X Invitrogen 1X 50ml
Glucose Sigma 10gm/liter 5gm
BSA (7.5%) Invitrogen 0.2% 13ml
10%NaN3 Sigma 0.02% 1ml
1M HEPES Sigma 5mM 2.5ml
Saponin Sigma 0.5% 0.25gm
Water Invitrogen 432
Triton X 100 SIgma 0.2% 1000ul

Wash MEDIA – 0.2% Triton X 100

Company Working conc Working conc
PBS-10X Invitrogen 1X 50ml
Glucose Sigma 10gm/liter 5gm
BSA (7.5%) Invitrogen 0.2% 13ml
10%NaN3 Sigma 0.02% 1ml
1M HEPES Sigma 5mM 2.5ml
Water Invitrogen 433

METHOD – LIVE STAINING METHOD

  1. Wash the cells with 1X PBS
  2. Add 1ml of hESC media and add 5ul of SeV antibody (1° Ab - Abcam chicken IgY Abcam 1:200 dilution) to the cells
    OR
  3. Add 1ml of hESC media and add 2ul of SeV antibody (1° Ab - rabbit SV antiserum -MBL – 1:500 dilution) to the cells
  4. Incubate cells for 1 hour at 37 degrees
  5. Wash cells 2-3X with DMEM or hESC media
  6. Add 1ml of hESC media and add 2ul of secondary anti-SeV antibody to the cells (1° Ab - Abcam chicken IgY Abcam OR donkey anti rabbit AlexaFluor 647)
  7. Incubate for 1 hour at 37 degrees
  8. Wash with DMEM media
  9. Add hESC media back to the wells
  10. Microscope

METHOD – FIXED STAINING METHOD

Using a 16% % paraformaldehyde, make working solution to 4% PFA in PBS. Make only what you require for the day. Fix the cells for 15’ in 4% Paraformaldehide/PBS at room temp.
- Take off the formaldehyde/PBS and wash 2x with PBS at RT with gentle rocking (do atleast once).
- Permeabilize the cells 5’ at RT with 0.2 % Triton-X-100 in saponin solution
- wash 2x with PBS at RT with rocking
- to block nonspecific antibody binding add minimum volume of saponin containing 10% secondary antibody serum for 60’ at RT (10% donkey serum / 10% goat serum etc.).
- Add primary antibody 1:200 dilution in blocking saponin solution O/N at 4 degrees.
- Aspirate off primary antibody and replace with saponin media for 5-10minutes with gentle rocking. Repeat twice, to give three washes in saponin media
- incubate 60’ with 100-200 ul of saponin media containing 1;200 dilution of secondary antibody (without secondary Ab serum).
-Remove secondary antibody and replace with 1 ml of saponin. Give three washes in saponin.
Do the last wash with Dapi (1:1000) dilution for 5-10minutes. Wash out Dapi using saponin solution (1-2X)
-Fluorescent microscope.

IMAGES
image

IMAGES
image

Troubleshooting:

If you overexpose, even negative images will appear as positive.

References:

Obtained protocols from Abcam and MBL websites.

Acknowledgements:

Obtained protocols from Abcam and MBL websites.

Comments (already in Stembook template):

***There should be an option to print the entire protocol, each section independently, or combinations of sections [1,3,4,5,6].

This page was last modified on October 18, 2012