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 |
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.
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 – 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
IMAGES
If you overexpose, even negative images will appear as positive.
Obtained protocols from Abcam and MBL websites.
Obtained protocols from Abcam and MBL websites.
***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