This is a four day procedure so it's best to start on Monday or Tuesday.

CASE SELECTION:

H&E stained thin sections are first reviewed by a pathologist, and areas of interest are outlined. Tissue blocks are then cut as follows:

3 consecutive 5-micron sections from a formalin fixed paraffin embedded block are cut and placed onto positively charged slides (some dissection protocols discourage use of charged slides). More can be cut if additional studies (e.g. immunohistochemistry) are being considered.

Stain the middle slide with H&E. Check that areas from the previous H&E slide are the same and if needed get the pathologist to review the new H&E slide (some small lesions will change diagnosis or even disappear in deeper sections). If the two H&E's look the same then Stain one or both with (see protocol below).

If the histopathology is at all difficult, photos are taken of areas to be dissected on both the methyl green and H&E slides. In general, the methyl green slide is photographed at a higher power than the H&E.

The pathologist then reviews the methyl green photo along side of the H&E slide. The methyl green photo is then marked by the pathologist to define the areas to be microdissected. If additional areas with no photos are suggested for use, mark the H&E slide, then take photos while dissecting to at least document what is taken. If there is any uncertainty, ask the Pathologist to re-review the area on the slide in question.

Day 1:

DISSECTION:

See photos of , , and .

Label the top of 0.5 ml pcr tubes, color code dot label them also.

Add 15 μl of 1X Buffer into each tube, close caps.

Do one case at a time. If normal is being dissected do this first.

Scrape away areas that you dont want away from the sample (use a #11 blade).

Take a new #15 blade and dip (don't dunk) tip of blade into the labeled tube with pcr pk buffer, put this very small drop from the tip of blade onto the sample area by tapping.

Start scraping tissue into the center of puddle. Watch puddle dry up to point where you can pick up the sample scrapings (do not overdry or tissue will fly away!!).

Place this carefully into the 0.5 ml pcr tube with the pcr/pk buffer.

The amount of pcr/pk buffer depends on the sample. In general it's 15 μl for anything less than 1 mm, and proportionally more if larger.

Go onto next area. Remember to use diiferent blades for each part of the tumor.

Overlay with 20 μl mineral oil. Close cap and seal with parafilm.

DIGESTION:

Incubate overnight for shaking at 120 rpm, 55℃.

Day 2:

Add 0.3 μl fresh conc (20 mg/ml stock) through the oil to sample. (It's 0.3 μl per 15ul original volume, so adjust as necessary).

Incubate overnight at 55℃.

Day 3:

(Repeat:) Add 0.3 μl fresh conc (20 mg/ml stock) through the oil to sample. (It's 0.3 μl per 15 μl original volume, so adjust as necessary).

Incubate overnight at 55℃.

Day 4:

Remove all parafilm from tubes. Inactivate Proteinase K for 10-15 minutes at 95 ℃ in PCR machine or hot water bath.

Remove oil by rolling samples and oil on parafilm and and pipette aqueous DNA into new tube.

SOLUTIONS

Digestion buffer: 1X PCR Buffer with 0.5% Tween20 / 0.4mg/ml pk stored in aliquots at -20 ℃.

PK: Stored as 20mg/ml aliquots at -20 ℃; can be refrozen a few times.

Methyl Green Staining

Methyl Green is used for staining of microdissected slides since we have found that hematoxylin (H&E) can interfere with PCR amplification. It is usually only necessary to stain and microdissect one slide, but two slides are used when the target is less than 1 mm in diameter.

A. Deparaffinization:

Xylene 2X 3 mins each.

100% ethanol 2 mins.

95% ethanol 2 mins.

70% ethanol 2 mins.

50% ethanol 2 mins.

dH₂O 2 mins.

B. Staining:

0.1-1% Methyl green (dip 3-6 times or leave ~15-60 seconds).

The section should not be too dark. The methyl green concentration can be varied if sections are generally too dark or too light. It's better to start with a lower concentration.

dH₂O 1-5 mins (depending on how blue/green the tissue is).

Air dry upright.

The slide is now ready for photographing and microdissection. It is better to use soon but can be stored for a few weeks if necessary.