Rhodamine-Conjugated Chitin Probe
From 2010.igem.org
(Difference between revisions)
(→Methanol Fixation) |
(→Methanol Fixation) |
||
Line 1: | Line 1: | ||
- | =Methanol Fixation= | + | ===Methanol Fixation=== |
# Rinse slide with ethanol and flame | # Rinse slide with ethanol and flame | ||
# Rinse slide with PBS and dry with KimWipe | # Rinse slide with PBS and dry with KimWipe | ||
Line 6: | Line 6: | ||
# Submerge in -20C absolute methanol for 5-10 min | # Submerge in -20C absolute methanol for 5-10 min | ||
# Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | # Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | ||
- | + | * '''Do not let cells dry for the rest of the procedure''' | |
- | ==Staining== | + | ===Staining=== |
# Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide) | # Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide) | ||
# Apply 50ul to the slide containing the sample | # Apply 50ul to the slide containing the sample | ||
# Allow stain to incubate overnight at RT in a dark room | # Allow stain to incubate overnight at RT in a dark room | ||
- | + | * ''To prevent stain from evaporating overnight'': Use a large glass petri dish. Soak a paper towel with PBS and place it at the bottom of the petri dish. Use 2 toothpicks to support the slide above the paper towel. This prevents any wicking action from occurring and pulling the stain off the slide. | |
# Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | # Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) |
Revision as of 19:30, 14 August 2010
Methanol Fixation
- Rinse slide with ethanol and flame
- Rinse slide with PBS and dry with KimWipe
- Apply cell sample to microscope slide and let air dry
- Submerge in -20C absolute methanol for 5-10 min
- Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)
* Do not let cells dry for the rest of the procedure
Staining
- Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide)
- Apply 50ul to the slide containing the sample
- Allow stain to incubate overnight at RT in a dark room
* To prevent stain from evaporating overnight: Use a large glass petri dish. Soak a paper towel with PBS and place it at the bottom of the petri dish. Use 2 toothpicks to support the slide above the paper towel. This prevents any wicking action from occurring and pulling the stain off the slide.
- Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)