5\. Laser Capture Microdissection (LCM) Of Gut Epithelial Cells and RT
PCR

Thaddeus Stappenbeck and Jeff Gordon

Department of Molecular Biology and Pharmacology

School of Medicine

Washington University

St. Louis, MO 63110

**Goals**

1\) Learn proper tissue procurement and handling for LCM.

2\) Learn and practice cutting and staining frozen sections for LCM.

3\) Gain hands on experience with LCM.

4\) Learn how to isolate and quantify RNA isolated from small quantities
of captured cells.

5\) Perform an assay to verify the presence of RNA in the sample
(RT-PCR).

**Freeze Adult Gut for LCM**

**Protocol**

1\) Excise tissues into ice-cold PBS. Work as quickly as possible!

2\) Flush the gut with ice-cold PBS by using 12-cc syringe and 1 1/2"
18-gauge blunt needle. Carefully hold end of intestine with forceps and
flush.

3\) Flush with OCT (Tissue Tek OCT 25608-930) with a blunt-gauge needle.

4\) Cut the gut into 2-cm segments.

5\) Place three adjacent segments in a cryomold (VWR: molds M7144-11
Tissue Tek) and overlay with additional OCT.

6\) Freeze tissue as follows:

> a\) In a styrofoam cooler, cool down 50-ml, side-arm flask, 10-cm
> glass petri dish with dry ice.
>
> b\) Spray Cryo-cool (Stephens Sci #48212-190) into flask to liquefy.
>
> c\) Pour liquid into the petri dish.
>
> d\) Submerge cryomold with gut sections completely under liquid
> cryocool, until bubbling ceases.
>
> e\) Leave in cooler surrounded by dry ice.

7\) Store at -80°C, wrapped in foil until sectioning.

**Preparation of Frozen Sections for LCM**

1\) Equilibrate blocks at -20°C for 20--30 min.

2\) Use gloves when handling slides, etc. to prevent RNase
contamination.

3\) Mount section on chuck in the cryostat and cut 5--7 μm sections.

> (Sections must be flat for LCM!)
>
> Can use polylysine-coated slides.

4\) Immediately place each section as cut into a slide holder (on dry
ice).

5\) Sections can be stored at --80°C for up to 1 week or used
immediately.

**Routine Staining of Frozen Tissue Sections for LCM**

Thaw 1 slide for 1 minute at room temperature (basically, time for frost
to melt from slide) or fix directly after cutting the section.

Fix in 70% ethanol for 15 sec at room temperature (use Falcon tubes and
RNase-free conditions).

Nuclease-free water dip 10x (Important! Must remove OCT completely)

Methyl green dip 1x (10 sec)

Nuclease-free water dip 10x

70% Ethanol dip 10x

95% Ethanol dip 10x

Eosin dip 1x (10 sec)

95% Ethanol dip 10x

95% Ethanol 1 min

100% Ethanol 1 min

100% Ethanol 1 min

Xylenes x3 5 min total

Air dry 2 min

An alternative stain is Nuclear Fast Red.

Fix in 70% ethanol for 15 sections at room temperature

Nuclease-free water dip 10x

NFR 10 seconds

Nuclease free water dip 10x

70% Ethanol dip 10x

95% Ethanol dip 10x

95% Ethanol dip 10x

95% Ethanol 1 min

100% Ethanol 1 min

100% Ethanol 1 min

Xylenes x3 5 min total

Air dry 2 min

Store in container with slide holder and dry-rite.

Reagents used:

Nuclease-free water and 10X PBS-Ambion

Methyl green and Nuclear Fast Red--Vector labs

Eosin-Richard Allen Scientific

Absolute ethanol--AAPER

Xylenes-use histology grade from Fisher UN1307

### LCM Tips 

### 

1.  Turn on laser, video monitor and controller.

2.  Load capsure cassette with 4 caps in holder.

3.  Turn on vacuum, place slide on stage with label over vacuum chuck
    hole.

4.  Center translation stage (important).

5.  Pick up cap (make sure cap is at the load line); place on slide to
    be dissected.

6.  Select a spot size and focus laser.

7.  Test laser over area with no tissue--should see proper wetting (if
    not, try flipping visualizer one time).

8.  After capture, cap reagent tube (use insertion tool).

**RNA Isolation: Stratagene Total RNA Microisolation Kit**

*Note for the purposes of the course (in the interest of time) the DNase
steps (8-12) can be cut out.*

1\) For each sample, add 1.5 μl BME to 200 μl denaturing solution (GITC)
in a 0.5-ml

Eppendorf tube (must be an Eppendorf brand tube!!!!!).

2\) Place cap with dissected cells on tube, invert for **5 min,** remove
cap.

3\) Add: 20 μl sodium acetate (2 M)

220 μl phenol

60 μl chloroform

**Ice for 10 min**

**Centrifuge 14,000 RRM, 30 min** **at 4°C**

4\) Transfer upper phase to new tube.

5\) Add: 2 μl glycocoblue (Ambion)

220 μl isopropanol

**Precipitate 10 min at -80°C**

**Centrifuge 14,000 RPM, 30 min 4**°**C**

6\) Wash pellet in 70% ethanol

7\) Air-dry pellet 10 min, re-suspend in 15.5 μl H~2~O

8\) Add: 2 μl 10x buffer (10 mM DTT, 100 mM MgCl~2~)

> 0.5 μl RNase inhibitor (Roche) 20 units total
>
> 2 μl DNase (Roche) 20 units total

**Incubate for 1 h 37**°**C**

9\) Add: 2 μl sodium acetate (2M)

> 22 μl phenol
>
> 6 μl chloroform

**Ice for 15 min, centrifuge 14,000 RPM, 30 min at 4**°**C**

10\) Collect upper phase.

11\) Add 23 μl isopropanol.

**-80**°**C for 10 min, centrifuge 14,000 RPM, 20 min at 4**°**C**

12\) Wash pellet 70% ethanol, air-dry pellet 10 min.

13\) Resuspend in 10 μl RNase-free water (store at -80°C).

**RNA Quantitation (continued)**

Quantify total RNA with Ribogreen (Molecular Probes)

Fluorescent plate reader- turn on 1/2 hr before use

Set parameters- filter select 2 (excite) and 2 (emit)

\- Menu-select wells to read (can read whole plate, rows, or columns)

Low range standard curve: dilute stock RNA 1:1000 (to 100 ng/ml);

add 1μl of diluted (to 10 μl) glycoblue per dilution

RNA H20 1:10 glycoblue 1:2000 ribogreen conc (ng/ml)

100 0 1 100 50

50 49 1 100 25

10 89 1 100 5

2 97 1 100 2

0 99 1 100 0

Dilute 1 μl of sample in 100 μl H20 100

**RT-PCR**

• Add 0.5 μl oligodT (250 ng/μl) to RNA from each sample

• Heat at 70°C for 5 min

• Cool to 42°C for 1 h

[-RT]{.underline} [+RT   ]{.underline}

dH20 12.4 μl 3.4 μl

RNase inhibitor (Roche) 0.6 μl 0.6 μl

5X buffer (Roche) 4 μl 4 μl

10 mM dNTPs 2 μl 2 μl

AMV (25 U/μl;Roche) 0 1 μl

Oligo annealed RNA sample 2 μl 8 μl

Incubate 1 h at 42°C.

[PCR]{.underline}

5 μl cDNA

2 μl 10X Taq buffer

0.4 μl 10 mM dNTPs

1 μl primer #1 (6 mM)

1 μl primer #2 (6 mM)

0.2 μl Taq polymerase

10.4 μl dH20

94°C for 1 min, 58°C for 1 min, 72°C for 2 min X 35 cycles

Run products on a 2% agarose gel.