6. Prepare a humidity chamber by laying parafilm on top of flat,
wet tissue paper. Cut two diagonal corners of the parafilm to
allow water to evaporate.
7. Lay the slides flat in the humidity chamber.
8. Incubate the slides in a 37
C hybridization oven overnight.
3.6 Posthybridization Washing
1. After hybridization, transfer the slides to a flat dish.
2. Float the hybridization membranes by adding 100 mL washing
buffer to the dish.
3. Wash the slides three times for 20 min with 100 mL washing
buffer.
4. Replace the washing buffer with 2Â SSC, and allow the slides
to equilibrate for 2 min.
3.7 Slide Mounting
1. Take the slides out from the washing buffer, and drain the
buffer on a paper towel.
2. Add three drops of mounting medium to each slide.
3. Place a clean cover glass over the samples. Lower carefully to
avoid bubbles.
4. Seal the sides of the cover glasses with clear nail polish.
5. Store the slides at 4
C until they are imaged.
3.8 Imaging
and Image Processing
Images should be captured using a confocal microscope with the
capability of spectral imaging and linear unmixing. We used a Zeiss
LSM 880 laser scanning confocal microscope with an Alpha PlanApochromat 100x/1.46 Oil DIC lens to acquire in situ images
using emission online fingerprinting mode (see Note 7).
1. Acquire a positive control emission spectrum using pure dye
with Lambda mode, in this case, AF594. Save this spectrum as
the positive control.
2. Acquire autofluorescence spectra using unlabeled sample with
Lambda mode. Save this spectrum as the autofluorescence.
3. At emission fingerprinting mode, assign detector RS1 and RS2
with positive control and autofluorescence, respectively. Then
perform a slow scan at laser speed 4.
4. Tile imaging can be applied for large samples. In this case, we
used a 2Â2 tile with 10% overlap.
5. After imaging, stitch the tiles with a 0.90 correlation threshold
using Zen software.
6. Filter the image using the 3, 3, 1 Median filter using Zen
software.
7. Adjust brightness and contrast equally for all images.
Single-Molecule FISH Imaging of RNAs in Plants
29
wet tissue paper. Cut two diagonal corners of the parafilm to
allow water to evaporate.
7. Lay the slides flat in the humidity chamber.
8. Incubate the slides in a 37
C hybridization oven overnight.
3.6 Posthybridization Washing
1. After hybridization, transfer the slides to a flat dish.
2. Float the hybridization membranes by adding 100 mL washing
buffer to the dish.
3. Wash the slides three times for 20 min with 100 mL washing
buffer.
4. Replace the washing buffer with 2Â SSC, and allow the slides
to equilibrate for 2 min.
3.7 Slide Mounting
1. Take the slides out from the washing buffer, and drain the
buffer on a paper towel.
2. Add three drops of mounting medium to each slide.
3. Place a clean cover glass over the samples. Lower carefully to
avoid bubbles.
4. Seal the sides of the cover glasses with clear nail polish.
5. Store the slides at 4
C until they are imaged.
3.8 Imaging
and Image Processing
Images should be captured using a confocal microscope with the
capability of spectral imaging and linear unmixing. We used a Zeiss
LSM 880 laser scanning confocal microscope with an Alpha PlanApochromat 100x/1.46 Oil DIC lens to acquire in situ images
using emission online fingerprinting mode (see Note 7).
1. Acquire a positive control emission spectrum using pure dye
with Lambda mode, in this case, AF594. Save this spectrum as
the positive control.
2. Acquire autofluorescence spectra using unlabeled sample with
Lambda mode. Save this spectrum as the autofluorescence.
3. At emission fingerprinting mode, assign detector RS1 and RS2
with positive control and autofluorescence, respectively. Then
perform a slow scan at laser speed 4.
4. Tile imaging can be applied for large samples. In this case, we
used a 2Â2 tile with 10% overlap.
5. After imaging, stitch the tiles with a 0.90 correlation threshold
using Zen software.
6. Filter the image using the 3, 3, 1 Median filter using Zen
software.
7. Adjust brightness and contrast equally for all images.
Single-Molecule FISH Imaging of RNAs in Plants
29