1. Harvest all tissues as quickly as possible, i.e., <4 min from time of killing.
2. Excise the abdominal–inguinal MG chain from one side of the animal and spread onto a pre-cut piece of transparency film, mark the lymph node chain on the bottom of the film using a permanent marker, place in a 4 × 6-in. heat seal bag (do not seal), and snap freeze in liquid nitrogen without embedding in OCT.
3. Excise tumors, place in cryovials, and snap freeze in liquid nitrogen.
4. Store tissues at -80°C until ready to cut frozen sections.
1. Clean the cryostat chamber, including the knife blade holder with 200 proof ethanol.
2. Remove a disposable microtome blade previously stored in 200 proof ethanol, allow to air dry, and insert into the cryostat knife blade holder.
3. Set cryostat to optimized temperature for tissue type and allow time to equilibrate, e.g., -24°C for mammary tumor or -30°C for MG.
4. Remove one tissue at a time from -80°C freezer, transport under dry ice, and place the sample in the cryostat.
5. MG: Remove film-mounted gland from bag, bisect the gland longitudinally through the marked lymph node chain using a clean scalpel or razor blade, then cut above the superior node and below the inferior node to obtain two halves of mammary tissue.
6. Using a room temperature object holder, dispense enough OCT on the holder to adequately cover the surface and place it on the cryobar to cool.
7. Once the OCT begins to turn opaque, place the sample on top of the OCT, dispense additional OCT to cover the sample, and place a heat extractor on top to flatten and freeze the OCT quickly.
8. Place the object holder in the chuck of the cryotome and rotate. MG tissue should be rotated with the edge of the lymph node chain angled to the knife blade.
9. Adjust cutting depth to 7 μm for tumor or 10 μm for MG.
10. Cut sections and place on clean, room temp, plain glass slides (orient sections in the middle toward the lower half of the slide).
11. Immediately place the slide on the Peltier cooled portion of the cryobar to freeze as quickly as possible.
12. Once frozen, place the slide in a pre-cooled, clean, small plastic slide box inside the cryo chamber. One box per sample.
13. Cut additional sections for use as replicates to minimize freeze/thaw problems, but limit total sectioning time to <20 min.
14. Return the sample and slide box of cut sections to -80°C until ready for dehydration and LCM.
1. Fill pre-cleaned (nuclease-free) plastic staining jars with Histogene® reagents and order as follows: 75% ethanol (EtOH), H20, 75% EtOH, 95% EtOH, 100% EtOH, xylene.
2. Remove one sample at a time from -80°C and transport under dry ice to the chemical fume hood.
3. Remove one slide from the slide box; do not thaw and immerse immediately in 75% EtOH for 30 s. Transfer the slide from one reagent to the next in the order listed above and incubate for 30 s in each reagent with the exception of xylene (1 min). A pair of forceps should be used to agitate the slide vertically in each reagent solution for the duration of each incubation period in order to ensure adequate reagent transfer. Regent should be drained from the slide prior to immersion in subsequent reagents to minimize cross-contamination. No more than four slides should be used in one set of reagents. The slide should be air-dried under the fume hood. Rapid reciprocal movement of the slide under the hood will help facilitate the air drying process.
4. Inspect the slide for any folds, wrinkles, or debris at the edges of the tissue section and remove with a clean scalpel blade.
5. Place a PrepStrip™ on top of the dried tissue section, rub index finger or thumb over the length of the strip three to four times, and peel the strip off of the section to remove any debris that might interfere with seating of the laser capture microdissection (LCM) cap.
6. Proceed immediately with LCM.
3.4 Laser capture microdissection
1. Load the dry slide in the LCM instrument, focus, and acquire a road map image of the tissue section.
2. Move the red box on the road map image to the desired location, right click, and choose "place cap at region center." The cap should be position such that only one half to three fourths of the cap surface covers the tissue section with the remainder covering a blank portion of the glass slide.
3. Using the ×10 objective, bring the tissue section into focus then move the active window to a blank portion of the cap.
4. Locate and focus the laser then test fire to verify adequate wetting (black ring with a clear center).
5. Default laser settings for pulse (1,500 μs), hits (1) and delay (0) should be adequate for 7-μm tumor sections, but the power level may need to be increased from 60 to 90 mW. Thicker MG sections (10 μm) may require increasing the number of hits from 2 to 20, and the delay should be increased from 0 to 10 μs.
6. Test fire the laser and set the spot size.
7. Draw a ROI on road map image and acquire the region as static image (no more than 30 image tiles).
8. Use a digital tablet in conjunction with the free-hand line and polygon tools to quickly draw or mark multiple areas on the static image intended for capture. When finished, right click and choose capture. Repeat the ROI selection and marking process on different areas within the cap as time permits.
9. Unload the cap and stamp three times on tacky portion of a clean self-adhesive note to remove debris.
10. Place the cap on a blank portion of the glass slide and reacquire the road map image.
11. Using the ×4 objective, draw a ROI around the cap and acquire as a static image. Inspect the image to verify successful capture of epithelial cells.
12. Open and pull the lid completely off of the microfuge tube filled with 30 μL of RNA extraction buffer (PicoPure® RNA isolation kit).
13. Remove the cap from the slide and seat the cap on the microfuge tube, being careful not to crack the tube.
14. Invert the tube and tap on the bench top several times to ensure the buffer covers the surface of the cap.
15. Total time from start of dehydration procedure to extraction buffer should be ≤30 min.
3.5 RNA Extraction
1. Place inverted tubes in a pre-warmed heating block and incubate at 42°C for 30 min.
2. Remove the tubes from the heating block and centrifuge at 800×g for 2 min.
3. Store tubes at -80°C or proceed with remainder of extraction procedure.
4. Pre-condition the RNA purification column using 250 μL of conditioning buffer and incubate for 5 min at room temp.
5. Centrifuge the column at 16,000×g for 1 min.
6. Pipette 30 μL of 70% EtOH into the cell extract from RNA extraction and mix by pipetting up and down; do not centrifuge.
7. Pipette the cell extract and EtOH mixture into the pre-conditioned purification column.
8. Centrifuge for 2 min at 100×g followed by centrifugation at 16,000×g for 30 s to bind RNA to the column.
9. Pipette 100 μL WB1 into the purification column and centrifuge for 1 min at 8,000×g.
10. DNase treatment (use RNase-Free DNase Set (Qiagen, catalog no. 79254).
11. Pipette 5 μL DNase I stock solution into 35 μL Buffer RDD (provided with RNase-Free DNase Set) and mix by gently inverting.
12. Pipette the 40 μL DNase incubation mix directly into the purification column membrane and incubate at room temperature for 15 min.
13. Pipette 40 μL PicoPure® RNA Kit WB1into the purification column membrane and centrifuge at 8,000×g for 15 s.
14. Pipette 100 μL (WB2) into the purification column and centrifuge for 1 min at 8,000×g.
15. Pipette another 100 μL WB2 into the purification column and centrifuge for 2 min at 16,000×g.
16. Check the purification column for any residual WB. If WB remains, re-centrifuge at 16,000×g for 1 min.
17. Transfer the column to a new 0.5 mL microcentrifuge tube provided in the kit.
18. Pipette 11 μL of elution buffer (EB) directly onto the membrane of the column and incubate for 1 min at room temperature.
19. Centrifuge the column for 1 min at 1,000×g to distribute EB in the column.
20. Centrifuge for 1 min at 16,000×g to elute RNA. The isolated RNA is now ready for use in downstream applications. The entire sample may be used immediately or stored at -80°C until ready to use.