Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Hello, My name is Dorte from Thea Hands Institute at the University of Technology, Reen Laser. Microdissection is a technique that allows to obtain a special cell population from very little amounts of parent tumor. The dissected cells can be used for transcriptomic and proteomic studies, DNA assessment or chromosome analysis.

Here we provide our protocol for acquiring human pancreatic meta cells from surgical specimens to be used for transcriptomic studies. Cut the pancreatic tissue into small pieces. Place one piece in the center of a cryo mold and cover it with Chilled embedding compound.

Freeze the tissue immediately With chilled two material Bhutan and stored at minus 80 degrees Celsius. Sectioning label 40 adhesive slides and pre-cool them inside the Christed Chamber. Fix the frozen tissue on the specimen holder with the OCT compound after trimming the cryo block.

Cut 40 10 micrometer thick sections. Transfer One section to one slide by touching the backside of the slide with your finger to warm only the position for placing the section on It.Try the Sections two to three minutes inside the cryos dead and store them afterwards at minus 80 degrees Celsius, prepare the Ation reagents and chill all liquids except seline on ice. This will increase the intrinsic autofluorescence of pet cells prior to the micro dissection.

Four sections will be derated each time. Handle two sections at once. Fix them first in 70%ethanol for 30 seconds.

Then wash the sections with five to six quick dips. In DPC treated water hereafter. Dehydrate the tissue one minute in 100%ethanol and repeat This step, incubate the slides In seline for four minutes.

Meanwhile, start the dehydration of another pair of sections. Finally, air dry the slides for three to five minutes In the dark. Protect the tissue from moisture and the autofluorescence from bleaching by placing the two slides back to back into a foil Wrap 50 ml Tube Before the section, clean the microscope with RNAs away and clamp the adhesive cap into the robo Mover.

Insert the slide Into the stage and move the cap closely over the section without touching it. Localize the eyelets by identifying the autofluorescent better cell areas. The intrinsic autofluorescence of human better cells appears yellow in the setup, whereas pancreatic ducts show bright green autofluorescence.

Select the better cell areas with the free hand selection tool and micro dissect them by Starting the laser. Dissect The eyelets of four derated cryo sections into one adhesive cap within 40 to 60 minutes. If desired, check the captured tissue by moving the cap to the checkpoint of the Microscope.

Remove the cap Out of the robo mover and pipe it. 10 microliter extraction buffer into the lid Of the cap for cell disruption. Incubate the cap upside down at 42 degrees Celsius for 30 minutes.

Spin down the lysate, label the tube and storage it at minus 80 decreased Celsius until the RNA extraction can occur. Perform the Ation laser, micro dissection and lysis with all 40 cryo dissects. Afterwards, unite the 10 lysates and continue with the RNA isolation using the UR PU RNA isolation kit following the supplied protocol to obtain 11 microliter RNA.

In general, We did not find a direct relationship between the amount of microdisect tissue and the RNA yield Differences in the time interval between the harvesting by the surgeons and the freezing of the pancreatic specimen after its examination by the pathologist, could account in part for therial quality and quantity of recovered RNA. Other key factors to consider are the laser energy applied for micro dissection with the lower energy, using the higher RNA integrity, as well as the laser focus and the distance of the laser pressure cutter pointing points. Additionally, the RNA quantity can be optimized by keeping the distance between the adhesive cap and the as low as possible.

Avoid loss of micro dissected tissue during the capturing process, provided that the laser micro dissection microscope is available. This procedure could be implemented at any research institutions performing partial pancreat ectomies, thereby increasing access to human eyelid material from both non-diabetic and diabetic subjects.