Troubleshooting Laser Capture Microdissection of *Porites compressa*

Troubleshooting Laser Capture Microdissection of Porites compressa

How to:

Notebook of steps taken and results

Monday, 4/27/26

  1. Sectioned POR_3 from the 3/10/26 fixation onto 2 RNAse & UV-treated PEN membrane slides exactly as written in the LCM Protocol
    1. Keep slide cold in the cryostat, briefly warm sections with finger on back of slide to adhere. Refreeze on cryostat plate.
    2. Once all sections are adhered, dry the slide at room temp in a sterile petri dish for 1 min
    3. Then, inside the dry ice cooler, pipette on ice-cold 100% ethanol and let sit 2 minutes
    4. Remove excess ethanol, gently blotting bottom of slide with kimpwipe but minimizing any lint contamination
    5. Transfer slide to falcon tube with dessicant, let sit out at room temp in this sealed tube for 15 minutes
    6. Bury sealed tube in dry ice and keep in dry ice until transfer to -80 ºC freezer.
  2. Also sectioned 3 glass slides for staining tests (different project)

Tuesday, 4/28/26

  1. Thawed and stained slides as written in the LCM Protocol
    1. Place slide on petri dish over dry ice
    2. Apply Cresyl violet staining solution (in 100% ethanol) directly with syringe and sterile filter to the section and incubate for 1 minute, swivel gently
    3. Rinse off stain by (pipetting) with ice-cold 70% ethanol
    4. And then place back on clean petri dish and cover slide with ice-cold 70% ethanol
      1. (ideally the ethanol just stays on the slide but if it rolls off then gently submerge the slide in 70% ethanol)
      2. Ideally this removes all the OCT. And hopefully no tissue.
      3. Be as gentle as possible and keep everything as cold as possible
    5. If any OCT remains, rinse or gently submerge slide in ice-cold RNAse-free water
      1. make sure this is not directly over dry ice but elevated, it will freeze if not
    6. Then submerge in or cover slide with ice-cold 100% ethanol for 1 minute to fully dry tissue and remove any excess water
    7. Air dry sample 1-2 in drying chamber with desiccant (or falcon tube with silica) or fume hood
      1. Proceed to LCM now, transport slide in falcon tube with silica packet

LCM Staining + Tissue Quality Assessment

Staining summary: Cresyl –> 70% EtOH –> RNase‑free water (brief) –> 100% EtOH

  1. Lots of purple stained OCT in tissue gaps, but tissue is overall looking okay for PEN membrane
  2. Pretty hard to distinguish oral epidermis and oral gastrodermis
  3. Slide 1 was better than Slide 2

Tubes collected: 1-13 (lost 6-8 in centrifuge…)

Data here: https://docs.google.com/spreadsheets/d/1iXE60arOpAE1sDYm0sR2PGPPH7mQKXzS5cIpavmqhCU/edit?usp=sharing

Example images:

Slide 1:

Slide 2:

Oral Epidermis Dissection Attempt Oral Gastrodermis Dissection Attempt
OE OG
OE OG

Monday, 5/4/26

  1. Sectioned POR_4 from the 3/10/26 fixation onto 2 RNAse & UV-treated PEN membrane slides but changed one variable:
    1. Slide 1: Normal protocol (air dry –> 100% ethanol –> 15 min air dry in falcon tube –> dry ice)
    2. Slide 2: Normal protocol (no ethanol –> 2 min air dry in falcon tube –> dry ice)

Wednesday, 5/6/26 - Ethanol‑Only Variant (No RNase‑free Water; Added 75% Pre‑Step)

  1. Thawed and stained slides with modifications to the LCM Protocol
    1. Place slide on petri dish; on tube rack over dry ice (not immediately on the dry ice otherwise the 70% ethanol can freeze)
    2. Attempt to remove OCT by submerging ice-cold 75% ethanol for 2 mins
    3. Apply Cresyl violet staining solution (in 100% ethanol) directly with syringe and sterile filter to the section and incubate for 1 minute, swivel gently
      1. Incubated for closer to 30 seconds
    4. Rinse off stain by (pipetting) with ice-cold 70% ethanol
    5. And then place back on clean petri dish and cover slide with ice-cold 70% ethanol
      1. (ideally the ethanol just stays on the slide but if it rolls off then gently submerge the slide in 70% ethanol)
      2. Ideally this removes all the OCT. And hopefully no tissue.
      3. Be as gentle as possible and keep everything as cold as possible
    6. If any OCT remains, rinse or gently submerge slide in ice-cold RNAse-free water
      1. make sure this is not directly over dry ice but elevated, it will freeze if not
    7. Then submerge in or cover slide with ice-cold 100% ethanol for 1 minute to fully dry tissue and remove any excess water
    8. Air dry sample 1-2 in drying chamber with desiccant (or falcon tube with silica) or fume hood
      1. Proceed to LCM now, transport slide in falcon tube with silica packet

LCM Staining + Tissue Quality Assessment

Staining summary: 75% EtOH –> Cresyl –> 70% EtOH –> 100% EtOH

  1. TONS of OCT remained. Wasn’t stained super purple but there were sheets and folds of OCT that hampered dissections a lot
    1. Likely because of removing the RNAse free water step?
  2. The slide that had the ethanol step at the cryostat (Slide 1) looked marginally better than Slide 2 in tissue quality
    1. But no difference in OCT amounts

Tubes collected: 14-24

Data here: https://docs.google.com/spreadsheets/d/1iXE60arOpAE1sDYm0sR2PGPPH7mQKXzS5cIpavmqhCU/edit?usp=sharing

Example images:

Slide 1:

Slide 2:

Oral Epidermis Dissection Attempt Oral Gastrodermis Dissection Attempt
OE OG
OE OG
OE OG

Wednesday, 5/6/26 PM

  1. Sectioned one more slide POR_4 from the 3/10/26 fixation onto 1 RNAse & UV-treated PEN membrane slide and mimicked the slide 2 change from 5/4/26:
    1. Slide 3: No ethanol protocol (2 min air dry in falcon tube –> dry ice)

Monday, 5/11/26 - High‑Aqueous / PAXgene‑Like Protocol

  1. Thawed and stained slides with modifications to the LCM Protocol
    1. Increased aqueous protocol with 75%, 50%, 30% EtOH and multiple H2O steps (based on Paxgene protocol)
      1. 1 min 75% EtOH
      2. 45 s 50% EtOH
      3. 30 s 30% EtOH
      4. 30 s RNAse free H2O
      5. Cresyl violet, 1 minute
      6. Dip in 75% ethanol
      7. Another RNAse free H2O dunk to remove remaining OCT
      8. 100% ethanol 1 minute

LCM Staining + Tissue Quality Assessment

Staining summary: 75% EtOH –> 50% EtOH –> 30% EtOH –> H2O –> Cresyl –> 75% EtOH –> H2O –> 100% EtOH

  1. Severe tissue loss. Too much water/washing.
    1. No ability to distinguish tissue layers
    2. When moving from the cold H2O to the cold 100% ethanol, i think the ethanol was SO cold that the water turned into ice in certain parts of the slide/tube, which looked not great
  2. Decided to still collect tissue for extraction testing, took big bulk pieces in different extraction buffers
    1. normal ProK digestion buffer from the LCM Protocol
    2. DNA/RNA shield
    3. RNA Lysis buffer from the extraction kit
  3. I also thawed Slide 2 from 4/28/26 which I didn’t collect any tissue from and did the same thing – collected cells in different buffers for testing (tubes 40-45)

Tubes collected: 31-39 & 40-45

Data here: https://docs.google.com/spreadsheets/d/1iXE60arOpAE1sDYm0sR2PGPPH7mQKXzS5cIpavmqhCU/edit?usp=sharing

Example images:

bulk

Monday, 5/11/26 PM - Ethanol-only staining attempt

  1. Sectioned from a new block (Time Series POR_R72_C2) onto 2 RNAse & UV-treated PEN membrane slide and mimicked the slide 2 change from 5/4/26:
    1. Slides 1&2: No ethanol protocol (2 min air dry in falcon tube –> dry ice)
  2. Stained Slide 1 right away
    1. 3 min 70% EtOH
    2. 2 min 70% EtOH
    3. 30s 96% EtOH
    4. 30s 100% EtOH
    5. Cresyl violet 1 minute
    6. 1 min 70% EtOH
    7. Extra rinse with 70% by pipetting to try to remove OCT
    8. 30s 96% EtOH
    9. 100% EtOH 1 minute
    10. Dried in fume hood

LCM Staining + Tissue Quality Assessment (5/13/26)

Staining summary: Cryostat (no overnight freeze) –> 70% EtOH –> 96% EtOH –> 100% EtOH –> Cresyl –> 70% EtOH –> 96% EtOH –> 100% EtOH

  1. Went to the scope on 5/11 but did not collect any samples. Collected samples on 5/13
  2. There was so much OCT, worse than 4/28
    1. Very purple and hard to distinguish tissues, but did some tests
  3. Although there appeared to be a lot of OCT and very dark stain that made seeing the tissues hard, the dissection/laser cutting was surprisingly easy compared to 5/6/26 for example

Tubes collected on 5/13/26: 60-63

Oral Epidermis Dissection Attempt Oral Gastrodermis Dissection Attempt
OE OG
OE OG

Wednesday, 5/13/26

  1. Thawed POR_R72_C2 Slide 2 and did wash steps to remove OCT only, no staining (all in petri dish over dry ice, all solutions ice cold)
    1. 3 min 70% EtOH
    2. 2 min 70% EtOH
    3. 10 s RNAse free H2O
    4. 30s 70% EtOH
    5. 30s 96% EtOH
    6. 1 min 100% EtOH
    7. Dried in fume hood

LCM Staining + Tissue Quality Assessment

Staining summary: No staining

  1. Honestly looks okay without the stain, but the stain helps a little bit to distinguish tissues and removing it clearly still didn’t solve all my problems
  2. There was still tissue loss, likely from the RNAse free water step

Tubes collected: 51-58

Data here: https://docs.google.com/spreadsheets/d/1iXE60arOpAE1sDYm0sR2PGPPH7mQKXzS5cIpavmqhCU/edit?usp=sharing

Example images:

Oral Epidermis Dissection Attempt Oral Gastrodermis Dissection Attempt
OE OG
OE OG

OG

Current thoughts

  • Original protocol of 70% EtOH + RNase‑free water + 100% EtOH gives the best OCT removal with tissue present but tissue quality still isn’t ideal
  • Ethanol‑only (75/96/100) is safer for tissue adhesion but leaves too much OCT to see tissue layers
  • PAXgene‑like (75/50/30 + water) protocol is really good for OCT removal but the tissue is horrendous

Next time I stain/section // for the Time Series if we feel okay moving forward:

  • Maybe we could move forward with the stain-less protocol and swap the RNAse free H2O step for a 50% ethanol step?
    1. 3 min 70% EtOH
    2. 2 min 70% EtOH
    3. 30 s 50% EtOH
    4. 30s 70% EtOH
    5. 30s 96% EtOH
    6. 1 min 100% EtOH
    7. Dried in fume hood
  • Or possibly add back in very light staining because it does help (like May 6)
    1. Cover with ice-cold 70% ethanol for 2-3 mins
    2. Replace with fresh ice-cold 70% ethanol for 2 mins
    3. Apply cresyl violet staining (in 100% ethanol), incubate 30 seconds
    4. Pour off excess stain
    5. Cover slide with ice-cold 70% ethanol, 30s
    6. Pour off excess stain
    7. Cover slide with fresh ice-cold 70% ethanol, 30s
    8. If any OCT remains, rinse or gently submerge slide in ice-cold RNAse-free water for 10s
    9. Dehydrate:
      1. 30s 70% EtOH
      2. 30s 96% EtOH
      3. 1 min 100% EtOH
      4. Dry in fume hood

RNA extraction results:

My RNA concentrations are much much lower than I am used to for LCM.

Extraction Protocol Here. I follow this closely, but there are some modifications I have tried in increase yield, noted in the table below:

  • Different cell collection buffers (my normal PK buffer, DNA/RNA shield, or RNA Lysis buffer)
  • Different digestion times
    • 15 min RT = 15 minutes room temperature
    • 15 min 56C 1400rpm = 15 minutes at 56 ºC on thermomixer 1400 rpm
    • 1 hr 56C 1400rpm = 1 hour at 56 ºC on thermomixer 1400 rpm
    • 1 hr 52C = 1 hour at 52 ºC in the Incubator Genie, gentle shaking
    • Overnight 56C = Overnight (5pm-10:30am) at 56 ºC in the Incubator Genie, no shaking

Best results seem to come from 1 hr 56C 1400rpm protocol.

Slide Section Tube # Number dissections Tissue Extraction Buffer Notes Total Dissection Area Digestion Tapestation assessment 1-3 TS Conc (pg/uL) pg in 8 uL pg/area
POR_3_Slide1 5 1 5 oral epidermis PK Buffer extracted 5/13/26 160,516 15 min RT 1 44.6 357 0.0022
POR_3_Slide1 5 2 5 oral gastrodermis PK Buffer extracted 5/13/26 86,158 1 hr 56C 1400rpm 1 54.9 439 0.0051
POR_3_Slide1 4 9 1 bulk - all tissues PK Buffer extracted 5/13/26 3,215,616 15 min RT 2 48.1 385 0.0001
POR_3_Slide1 1 13 2 bulk - all tissues PK Buffer extracted 5/13/26 6,681,317 1 hr 56C 1400rpm 3 108.0 864 0.0001
POR_4_Slide1 1 17 3 oral epidermis PK Buffer extracted 5/8/26 PM 234,535 1 hr 52C 1 33.0 264 0.0011
POR_4_Slide1 1 18 3 oral gastrodermis PK Buffer extracted 5/8/26 PM 183,347 1 hr 52C 1 33.9 271 0.0015
POR_4_Slide1 4 21 5 oral epidermis PK Buffer extracted 5/8/26 AM 346,744 15 min RT 1 43.5 348 0.0010
POR_4_Slide1 4 22 6 oral gastrodermis PK Buffer extracted 5/8/26 AM 303,230 15 min RT 1 44.7 358 0.0012
POR_4_Slide1 5 23 1 bulk - all tissues PK Buffer extracted 5/8/26 PM 830,657 1 hr 52C 2 33.1 265 0.0003
POR_4_Slide1 5 24 1 bulk - all tissues PK Buffer extracted 5/8/26 AM 1,002,718 15 min RT 2 38.3 306 0.0003
POR_4_Slide3 1 31 1 bulk DNA/RNA Shield extracted 5/13/26 2,401,591 15 min RT 1 46.3 370 0.0002
POR_4_Slide3 2 32 1 bulk DNA/RNA Shield extracted 5/13/26 2,575,547 15 min 56C 1400rpm 2 46.1 369 0.0001
POR_4_Slide3 4 33 1 bulk RNA Lysis Buffer extracted 5/13/26 1,861,138 15 min RT 1 48.4 387 0.0002
POR_4_Slide3 5 34 1 bulk PK Buffer extracted 5/13/26 2,534,590 15 min 56C 1400rpm 3 65.9 527 0.0002
POR_R72_C2_2_unstained 2 51 5 oral epidermis PK Buffer extracted 5/14/26 110,765 Overnight 56C 1 31.4 251 0.0023
POR_R72_C2_2_unstained 2 52 5 oral gastrodermis PK Buffer extracted 5/14/26 84,931 1 hr 56C 1400rpm 1 27.1 217 0.0026
POR_R72_C2_2_unstained 3 55 6 oral epidermis PK Buffer extracted 5/14/26 276,815 1 hr 56C 1400rpm 2 24.4 195 0.0007
POR_R72_C2_1 2 60 5 oral epidermis PK Buffer extracted 5/14/26 343,268 1 hr 56C 1400rpm 1 35.4 283 0.0008
POR_R72_C2_1 2 61 5 oral gastrodermis PK Buffer extracted 5/14/26 239,654 15 min 56C 1400rpm 2 26.5 212 0.0009
   
2026-05-08-AM.png 17, 18, 21
2026-05-08-PM.png 22, 23, 24
2026-05-13.png 1, 2, 9, 13, 31, 32, 33, 34
2026-05-14.png 51, 52, 55, 60, 61

RNA Library Prep:

As noted above, my RNA concentrations are much much lower than I am used to for LCM. But I still want to test these samples to see what we can get. So started library prep on 5/14/26 for the following samples:

Slide Section Tube # Number dissections Tissue Extraction Buffer Notes Total Dissection Area Digestion Tapestation assessment 1-3 TS Conc (pg/uL) pg in 8 uL pg/area
POR_3_Slide1 1 13 2 bulk - all tissues PK Buffer extracted 5/13/26 6,681,317 1 hr 56C 1400rpm 3 108.0 864 0.0001
POR_4_Slide3 5 34 1 bulk PK Buffer extracted 5/13/26 2,534,590 15 min 56C 1400rpm 3 65.9 527 0.0002
POR_R72_C2_1 2 61 5 oral gastrodermis PK Buffer extracted 5/14/26 239,654 15 min 56C 1400rpm 2.0 26.5 212 0.0009

Library Prep Protocol Here

5/15/26 cDNA Results

I used 20 cycles for cDNA amplification (Step 2.4.3). They look pretty great!

cDNA-all.png

cDNA_13.png

cDNA_34.png

cDNA_61.png

cDNA and planned cycles/indeces

Sample Type cDNA concentration amount cDNA input (conc * 26 uL) PCR cycles for final step Index_ID index
#13 2.26 58.76   E7500S-18 GTCCGC
#34 3.09 80.34   E7500S-22 CGTACG
#61 3.05 79.30   E7500S-23 GAGTGG

Full results can be found here.

5/15/26 Final Library Results

I used 8 cycles for Final Library amplification (Step 2.10.3). They look amazing!!

lib-all.png

lib_13.png

lib_34.png

lib_61.png

Full results can be found here.

Pooling

Submitting a pool of 50 uL at 4 nM concentration.

Written on April 25, 2026