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Template:Team:ATOMS-Turkiye/Lab:Protocols:Immuno
From 2013.igem.org
(Difference between revisions)
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=IMMUNOFLUORESCENCE = | =IMMUNOFLUORESCENCE = | ||
| - | Aspirate the medium from wells. Wash wells with PBS 1000 µl/well. | + | * Aspirate the medium from wells. Wash wells with PBS 1000 µl/well. |
| - | Fix cells with prewarmed 4% Paraformaldehyde/PBS | + | * Fix cells with prewarmed 4% Paraformaldehyde/PBS |
| - | 500 µl /well for 15 min.at RT (add very slowly). | + | * 500 µl /well for 15 min.at RT (add very slowly). |
| - | Remove supernatant. | + | * Remove supernatant. |
| - | Treat the cells for 15 min. at RT with prewarmed (37oC) TZN buffer 500 µl/well (add very slowly). Slowly mix on shaker. | + | * Treat the cells for 15 min. at RT with prewarmed (37oC) TZN buffer 500 µl/well (add very slowly). Slowly mix on shaker. |
| - | Aspirate supernatant. | + | * Aspirate supernatant. |
| - | Wash wells with PBS, 750 µl/well, 5 min X 3 on shaker. | + | * Wash wells with PBS, 750 µl/well, 5 min X 3 on shaker. |
| - | Add Blocking Solution 500 µl/well, incubate for 1 hr at RT. Mix slowly on shaker. | + | * Add Blocking Solution 500 µl/well, incubate for 1 hr at RT. Mix slowly on shaker. |
| Line 18: | Line 18: | ||
| - | Aspirate off the blocking solution. | + | * Aspirate off the blocking solution. |
| - | Add 100 µl/ well 1st Ab. mixture. | + | * Add 100 µl/ well 1st Ab. mixture. |
| - | Seal the plate with parafilm, incubate at RT for 2 hr or o/n @ 4oC. | + | * Seal the plate with parafilm, incubate at RT for 2 hr or o/n @ 4oC. |
| - | Wash with PBS-TX 0.3%, 750 µl/well, 5 min X 1 on rocking shaker. | + | * Wash with PBS-TX 0.3%, 750 µl/well, 5 min X 1 on rocking shaker. |
| - | Wash with PBS 750 µl/well, 5 min X 2 on rocking shaker. | + | * Wash with PBS 750 µl/well, 5 min X 2 on rocking shaker. |
| - | Add 100µl/ well 2nd Ab. Work in dark from this point! | + | * Add 100µl/ well 2nd Ab. Work in dark from this point! |
| - | Incubate the plate at RT for 1 hr. | + | * Incubate the plate at RT for 1 hr. |
| - | Wash with PBS 1ml/ well , 5 min X 3 on rocking shaker. | + | * Wash with PBS 1ml/ well , 5 min X 3 on rocking shaker. |
| - | Add 300 µl/well TO-PRO-3 (1 µM) (light sensitive!) | + | * Add 300 µl/well TO-PRO-3 (1 µM) (light sensitive!) |
| - | Incubate at RT for 15 min. | + | * Incubate at RT for 15 min. |
| - | Wash with PBS 1 ml/well, 5 min X 3 on rocking shaker. | + | * Wash with PBS 1 ml/well, 5 min X 3 on rocking shaker. |
| - | Add 1 drop of Prolong Gold Mounting Medium onto slides for each coverslip. | + | * Add 1 drop of Prolong Gold Mounting Medium onto slides for each coverslip. |
| - | Take out coverslip from the well. Invert and put on mounting soln. on the slide. Seal | + | * Take out coverslip from the well. Invert and put on mounting soln. on the slide. Seal |
| - | coverslip with nail polish. | + | * coverslip with nail polish. |
| - | Let the coverslip dry. Slides can be stored at 4oC for a long time (at dark). | + | * Let the coverslip dry. Slides can be stored at 4oC for a long time (at dark). |
| - | Take images with confocal microscope. | + | * Take images with confocal microscope. |
Latest revision as of 02:35, 5 October 2013
IMMUNOFLUORESCENCE
- Aspirate the medium from wells. Wash wells with PBS 1000 µl/well.
- Fix cells with prewarmed 4% Paraformaldehyde/PBS
- 500 µl /well for 15 min.at RT (add very slowly).
- Remove supernatant.
- Treat the cells for 15 min. at RT with prewarmed (37oC) TZN buffer 500 µl/well (add very slowly). Slowly mix on shaker.
- Aspirate supernatant.
- Wash wells with PBS, 750 µl/well, 5 min X 3 on shaker.
- Add Blocking Solution 500 µl/well, incubate for 1 hr at RT. Mix slowly on shaker.
Blocking Solution (f)
NGS 10%
BSA (10%) 1%
PBS-Tx 0.3%
- Aspirate off the blocking solution.
- Add 100 µl/ well 1st Ab. mixture.
- Seal the plate with parafilm, incubate at RT for 2 hr or o/n @ 4oC.
- Wash with PBS-TX 0.3%, 750 µl/well, 5 min X 1 on rocking shaker.
- Wash with PBS 750 µl/well, 5 min X 2 on rocking shaker.
- Add 100µl/ well 2nd Ab. Work in dark from this point!
- Incubate the plate at RT for 1 hr.
- Wash with PBS 1ml/ well , 5 min X 3 on rocking shaker.
- Add 300 µl/well TO-PRO-3 (1 µM) (light sensitive!)
- Incubate at RT for 15 min.
- Wash with PBS 1 ml/well, 5 min X 3 on rocking shaker.
- Add 1 drop of Prolong Gold Mounting Medium onto slides for each coverslip.
- Take out coverslip from the well. Invert and put on mounting soln. on the slide. Seal
- coverslip with nail polish.
- Let the coverslip dry. Slides can be stored at 4oC for a long time (at dark).
- Take images with confocal microscope.
