LCM Sample Preparation:

staining

Pat Stockton and Yelena Golubeva

2

Fixative and Stain Requirements.

1. Compatibility with LCM (preserves the integrity of extracted molecules; doesn't deplete quantity).

2. Morphological details on LCM screen.

3. Good for different tissues.

4. Good cells pick-up.

5. Fixatives: alcohol/glacial acetic acid, 70%, 75%, 95% alcohol, methanol, acetone.

6. Stains: light staining and fast (one-step stain better).

LCM Stains

1. Arcturus

HistoGene™LCM frozen

section staining kit

2. Ambion LCM staining kit

3. Arcturus HistoGene™

Immunofluorescence

staining kit

4. Other stains:

Methyl green, Toluidine

blue, H&E, IHC

Arcturus

Ambion

In-house H&E

Stain appearance on LCM screen

One step cresyl-eosin (human kidney tumor,

OCT section)

Cresyl violet (mouse brain tumor,

tumor, paraffin section)

Mayer’s Hematoxylin (mouse mammary gland duct

epithelium, OCT section)

Cresyl violet (human skin melanoma, archival

paraffin section)

Staining Test

H&E Cresyl-eosin

Cresyl with methylgreenCresyl

Critical Points of the LCM Staining

Protocol

1. RNAse free procedure and reagents (test for RNA quality in your set-up)

2. Exact timing of staining steps

3. Water- push it out of the protocol!

4. How many slides to stain at once?

5. Slide storage in xylene and in a desiccator

H&E Stain for Frozen LCM Section.RNase-free conditions and reagents for the whole procedure. Staining is performed in 50 ml Falcon tubes

filled with 25 ml of required reagent.

• Quickly move a frozen slide from dry ice to 70% ethanol (-20ºC) inside the cryobox and incubate for 30

seconds at RT.

• Apply 900 µl of RNase-free water to the section for 5 seconds immediately drain the slide by touching a Kim

wipe with the edge of the slide.

• Apply a maximum of 200 µl of Hematoxylin-2 to the section, immediately drain the slide by touching a

kimwipe with the edge of the slide.

• Apply 900 µl aliquot of Bluing reagent for 5 seconds and immediately drain the slide by touching a kimwipe

with the edge of the slide.

• Place the slide in 70% ethanol 5 seconds

• Apply a maximum of 200 µl of eosin Y (diluted 1:10) to the section and immediately place the slide in 100%

ethanol (#1) for 5 seconds.

• Transfer the slide in 100% ethanol (#2), invert the tube once and incubate for 30 seconds

• Transfer the slide in 100% ethanol (#3) for 30 seconds

• Transfer the slide in xylene (#1), invert the tube once and incubate for 2 minutes.

• Transfer the slide in xylene (#2), invert the tube once and incubate for 3 minutes.

• Air dry the slide for 5 minutes in a fume hood.

• The slide is ready for LCM.

Note: don’t invert the tube in case of a section poor adherence to the slide/membrane

One-step Crezyl Violet/ EosinY Stain for Frozen LCM

Section.

RNase-free conditions and reagents for the whole procedure. Staining is performed in 50 ml Falcon tubes

with 25 ml of required reagent.

1. Prepare fresh staining mixture for 4 slides: 200µl of crezyl violet acetate stock (dissolve 250 mg of crezyl

violet in 25 ml of 100% ethanol, mix on a shaker overnight, filter for RNAse-free conditions, store at +4ºC),

200µl eosin Y and 400µl of Rnase-free water, vortex at high for 30 seconds, centrifuge at 6000 rpm for 1

minute, pipette out from the surface.

2. Prepare fresh fixative: to 25 ml of 100% ethanol add 750µl of glacial acetic acid and mix thoroughly.

3. Quickly move a frozen slide from dry ice to the tube with fixative (-20ºC) inside the cryobox and incubate

for 30 seconds at RT.

4. Apply 900µl of RNAse-free water to the section for 10 seconds, and immediately drain the slide by touching

a kimwipe with the edge of the slide.

5. Apply a maximum of 200µl of staining mixture to the section for 5 seconds, immediately drain the slide by

touching a kimwipe with the edge of the slide.

6. Place the slide in 100% ethanol (#1) for 5 seconds.

7. Transfer the slide in 100% ethanol (#2), invert the tube once and incubate for 30 seconds.

8. Transfer the slide in xylene (#1), invert the tube once and incubate for 2 minutes.

9. Transfer the slide in xylene (#2), invert the tube once and incubate for 3 minutes.

10. Air dry the slide for 5 minutes in a fume hood

11. The slide is ready for LCM.

Note: don’t invert the tube in case of a section poor adherence to the

slide/membrane

One-step Crezyl Violet (in 100%ETOH) Stain for

Frozen LCM Section.

RNase-free conditions and reagents for the whole procedure. Staining is performed in 50 ml Falcon tubes with 25 ml of

required reagent.

1. Prepare the stain: dissolve 250 mg of crezyl violet acetate in 25 ml of 100% ethanol, mix on a shaker overnight,

filter for RNAse-free conditions, store at +4ºC. ). Take the required aliquot out of refrigerator, vortex at high for 30

seconds, centrifuge at 6000 rpm for 1 minute, pipette out from the surface.

2. Prepare fresh fixative: to 25 ml of 100% ethanol add 750µl of glacial acetic acid and mix thoroughly.

3. Quickly move a frozen slide from dry ice to the tube with fixative (-20ºC) inside the cryobox and incubate for 30

seconds at RT.

4. Apply 900µl of RNAse-free water to the section for 10 seconds, and immediately drain the slide by touching a

kimwipe with the edge of the slide.

5. Apply a maximum of 200µl of staining mixture to the section for 5 seconds, immediately drain the slide by

touching a kimwipe with the edge of the slide.

6. Rinse the slide in 100% ethanol (#1) for 5 seconds.

7. Transfer the slide in 100% ethanol (#2), invert the tube once and incubate for 30 seconds.

8. Transfer the slide in 100% ethanol (#3), and incubate for 30 seconds.

9. Transfer the slide in xylene (#1), invert the tube once and incubate for 2 minutes.

10. Transfer the slide in xylene (#2), invert the tube once and incubate for 3 minutes.

11. Air dry the slide for 5 minutes in a fume hood

12. The slide is ready for LCM.

Note: don’t invert the tube in case of a section poor adherence to the slide/membrane.

Cresyl Violet Acetate (in nuclease-free water) Stain for Frozen LCM Section

Reagents.

Fixation

OCT Removal (1)

OCT Removal (2)

OCT Removal (3)

Stain application

Rinse in ethanol.

Dehydration

Clearing in Xylene

Slide drying

Move a dry slide into a desiccator for 10’ to improve dissection