Laser Capture Microdissection of Paraformaldehyde-Fixed Mouse Liver Tissue for RNA Analysis

Our research mainly investigates hepatocytes-related pathophysiology in the context of chronic liver diseases. This protocol enables cell isolation in liver tissue slices and RNA extraction. Unlike FFPE, or Paraformaldehyde-fixed or CT protocol preserve and itegrated, enabling precise micro discussion and high quality samples for downstream OMI analysis.

To begin expose pen membrane slides to ultraviolet light four 30 minutes. Coat the slides with 0.1 milligram per milliliter of poly L lysine and allow them to air dry. Seal the edges of each slide with a neutral mounting medium.

Using an RNase decontamination agent, wipe all accessible surfaces of the cryostat and all tools. Using 75%ethanol. Clean the brush and the tools thoroughly.

Now, set the cryostat temperature to minus 20 degrees Celsius and allow it to stabilize. Retrieve the liver samples from minus 80 degrees Celsius storage. Place the samples into the cryostat chamber for at least 15 minutes to equilibrate.

Apply a two to three millimeter thick layer of OCT compound onto the specimen stage. Then place the specimen stage on a quick free shelf or inside the cryostat chamber to pre-solidify the compound layer. After applying a small amount of OCT compound onto the pre-solidified layer, place the equilibrated tissue block horizontally onto the compound and hold it in place for 10 to 15 seconds.

Then mount the specimen stage with the attached tissue block onto the cryostat specimen head. Now insert the blade carefully between the clamping plates and back plates from one side of the cryostat. Clamp the clamping lever to secure the blade.

Adjust the cryostat thickness setting to 10 micrometers. Cut the tissue into sections and mount them directly onto the pretreated pen membrane slides inside the cryostat chamber, maintained at minus 20 degrees Celsius. Thaw the slides at room temperature for 20 minutes.

Wash the slides three times with RN ACE free PBS four 10 seconds each wash. Add filtered Mayer's hematoxylin solution onto the tissue sections for five to 10 seconds. Then wash the slides three times with ribonuclease free water for 10 seconds each wash to remove residual hematoxylin.

Assess the staining quality under a microscope. Using absorbent paper, blot the slides dry and let them air dry completely. Wipe the laboratory bench surface with RNase decontamination agent.

Turn on the controller and allow the microscope to complete initialization. Rotate the key clockwise from the off position to the on position to initiate the laser system. Turn on the computer and launch the laser capture micro dissection software.

Then wait for initialization to complete. Now insert a sterile 0.2 milliliter PCR tube into the collection device. Using a pipette add 10 microliters of protease K digestion buffer to the cap of the PCR tube.

Load the collection device into the microscope specimen holder. In the change collector device window. Go to adjust reference point, adjust the camera focus to clearly view the reference point and use the arrow controls to center it if necessary.

Load a tissue section into the specimen holder with the tissue facing down and the label position to the right. Select the 10 x objective lens and identify regions of interest with specific pathological features. Navigate to the laser menu.

Click calibrate, select yes and complete calibration. In the laser control panel, set laser power to 50, aperture to five and speed to nine. Then use the graphic toolbar to draw a circle around the target area.

Select single shape, press start cut to perform laser capture micro dissection and position the collection device to collect the dissected tissue. To capture documentation, images, select file and choose save images to save before and after images in the preferred format. After collecting the desired cells, click unload to remove the collector from the stage.

Verify that 10 microliters of protease K digestion buffer remains in the cap and replenish if necessary. Close the tube and vortex it thoroughly. Then spin the tube briefly in a centrifuge.

Store the tube at minus 80 degrees Celsius until RNA extraction. The hematoxylin-stained liver section on the pen slide was visualized under the microscope before and after laser capture microdissection. Regions of interest exhibiting micro vesicular steatosis were precisely collected into the cap of A PCR tube.

Preserving intact cellular morphology. Total ribonucleic acid extracted from approximately 1000 and 2000 laser capture microdissection isolated cells yielded 110.7 nanograms and 213.3 nanograms respectively. All samples showed high purity with absorbance of 260 by 280 nanometer ratios between 1.9 and 2.1.

Gel electrophoresis revealed distinct 28 s and 18 s ribosomal ribo nucleic acid bands. Bioanalyzer profiles of ribonucleic acid from as few as 1000 cells. Gave a ribonucleic acid integrity number of 6.7.

Q-R-T-P-C-R for the housekeeping gene Beta actin yielded cycle threshold values ranging from 17 to 19. Bioanalyzer assessment of sequencing libraries constructed from 50 nanograms of total ribonucleic acid showed a characteristic peak between 300 and 500 base pairs. RNA sequencing of these libraries yielded high quality data with both Q 20 and Q 30 scores, exceeding 90%and a low error rate across replicates.

An important consideration is preventing anti degradation via control cessation, rapid staining and straight conditions through our processing. Ordering this procedure and it can be used for real time PCR and a sequencing with fragmentation optimized library keys. Established in liver samples.

This protocol enables hepatocyte subgroup profiling and supports pathology focused hepatology research.