In-situ Hybridization (ISH) – RLOv Membrane Gene 1, Tail Fiber Gene: Day 3

All washes/rinses were performed in cylindrical glass slide incubators at room temp (30mL):

  • Slides were briefly rinsed in dH2O three times.
  • Slides were counter stained with 0.05% aqueous Bismark Brown Y for 3mins.
  • Slides were briefly rinsed in dH2O, then 70% EtOH, then 100% EtOH.
  • Slides were air-dried in the fume hood.
  • Coverslips were added to each slide with three drops of Permount.
  • Permount was allowed to dry O/N at RT.

Images were captured using Nikon BR Essentials.

The same section of each slide (within an accession number set) was captured at 4x, 10x, and 20x magnifications for comparisons. Auto white balance adjustment was the only image manipulation performed. All images (see Results below) are as they were captured by the software.

Results:

Quick summary: Both probes appear to be functional! With that being the case, I will proceed to run ISH on black abalone samples (these test ISHs were with red abalone) for the proper assessment of RLOv localization.

All images are here (Dropbox): 20151204_ISH_RLOv

Tryptic images of 10x magnifications are presented below showing the H&E staining, negative control and RLOv probe.

ISH staining is expected to appear as dark brown staining.


08:1-7 (RLOv)

H&E – RLO inclusions are seen as the deep purple oblong structures.

Negative Control – RLO inclusions exhibit no staining and appear as oblong empty regions. These regions also no have any apparent cell wall/membrane around them. This is in contrast to the two other accession groups (08:1-12 & 08:1-15).

RLOv tail fiber – Staining is noticeable surrounding the RLO inclusion locations, but not within the inclusions. The staining is similar to the 08:1-12 negative control. So, it’s difficult to say if the staining in this sample is binding to its intended target (RLOv tail fiber) or if the difference seen is simply due to the particular section of this tissue.

RLOv membrane gene 1 – Not shown due to this region of tissue not being present on the slide.

 


 

08:1-12 (RLO CLASSIC)

H&E – RLO inclusions are seen as the deep purple oblong structures.

Negative Control – RLO inclusions appear similar to air bubbles, with no staining within them.

RLOv probes – Both probes stain within the RLO inclusions.


 

08:1-15 (RLO STIPPLED)

H&E – RLO inclusions are seen as light purple bulbous structures.

Negative Control – RLO inclusions are actually stained brown and are very noticeable. This is not expected.

RLOv membrane gene 1 - The staining is in the same locations as the negative control RLO inclusions. Intensity-wise, the staining seen from this probe is not much different than the negative control. However, the way the staining appears within the inclusions is different than the negative control. Not sure if this indicative of the probe working or if the different appearance is due to difference in tissue sections.

RLOv tail fiber – The staining is in the same locations as the negative control RLO inclusions. However, the intensity of the staining with the RLOv tail fiber probe is a much deeper brown, suggesting that the probe is binding within the RLO inclusions.

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