Tag Archives: histology

Sample Annotation – Olympia oyster histology blocks (from Laura Spencer)

I’ve been asked to isolate RNA from some paraffin-embedded Olympia oyster gonad tissue.

Despite some excellent documentation by Laura Spencer (images of tissue layouts in histology cassettes and a corresponding cassette mapping key file), the histology facility seems to have flipped some things around and/or repositioned/split the contents of each cassette. This makes ID-ing the proper tissues tedious and, at times, difficult.

The list of tissues that needs to be processed is listed in this GitHub Issue #648. I’ve also added the list below:

NF-10 22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

Prior to beginning RNA isolations, I have annotated images of the histology blocks and will be waiting for Laura to confirm that my annotations are correct. I will be posting a link to this notebook entry in the GitHub issue listed above for her to view and wait for her confirmation.

UPDATE 201700707 – Laura has indicated that many of my annotations are incorrect. Katie has gone through and made proper identification: https://github.com/sr320/LabDocs/issues/648#issuecomment-313792588

 

Additionally, as indicated in the GitHub Issue above, histology block “Oly 14″ does not have a corresponding tissue cassette photo (containing sample NF-10 26). Without the original image, I don’t think I can make an accurate guess on how the tissues are oriented in the resulting two histo blocks (see below).

 

BLOCKS 5

 

BLOCK 6

 

 

BLOCK 9

 

 

BLOCK 10

 

 

BLOCKS 14 (unable to annotate at time of posting)

 

 

 

BLOCK 15

 

 

 

BLOCK 21

 

 

 

 

BLOCK 22

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Samples Received – Green Abalone Slides

Received green abalone (Haliotis fulgens) slides from Fiorenza Micheli (Hopkins Marine Station, Stanford University). These are related to the tissue she sent to us on 20160519.

Unfortunately, the writing on the casettes that were submitted to DPMG came off during processing (see note on form below). As such, the facility was unable to identify which slides came from which histology blocks. Not sure what we’ll be able to do with these. The box of slides has been stored in FSH 236 with other slide boxes.

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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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Abalone Sampling – Post-esophageal & Digestive Gland Tissues

Ava sent up abalone for sampling from California for tissue sampling. Here’s the summary of how things went.

  • Animal temps @ -8.0C when opened at lab
  •  ~120 animals sampled
  •  Only 1 dead animal found- Additional dead abalone included in shipment were NOT sampled; too dead
  • Started @ ~11AM, finished at 5:15PM (3 people dissecting, 1 person weighing)
  • All mesh bags were saved and are in a 10% bleach solution over night in sink in 236
  • Histo cassettes put in Davidson’s over night; need to be transferred to 70% EtOH tomorrow
  • Histo cassette layout: animal #1 upper left, animal #2 upper right, animal #3 lower left
  • Tubes for 15:9 sampling have labels applied
  • Histo cassettes for 15:9 sampling have NOT been labelled
  • Empty shells have been labelled and saved; will need to be weighed/measured later

 

 

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RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

Isolated RNA from geoduck gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Grace’s notebook for full details on samples and preservation.

 

RNA was isolated from only two samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks to test out the kit:

  • 34
  • 42

IMPORTANT:

  • Prior to beginning, I prepared Buffer TR1 by adding 10μL of β-mercaptoethanol (β-ME) to 1000μL of Buffer TR1). This will be good for up to six weeks at RT.
  • Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.

Five 5μm sections were taken from each block.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 19,000g.
  • Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
  • Samples were eluted with 34μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Samples were stored at -80C in Shellfish RNA Box #5.

NOTE: The spreadsheet linked indicates other samples exist in the slots that I placed these two samples. Will need to update the spreadsheet to be accurate.

Results:

 

 

Looks like the kit worked! Yields are pretty good (~800ng) from each. The 260/280 ratios are great for both samples. Oddly, the 260/230 ratios for the two samples are pretty much polar opposites of each other; not sure why.

Will proceed with the remainder of the samples that were selected by Steven and Brent. Or, maybe I should try to make some cDNA from these RNA samples to verify the integrity of the RNA…

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