The overall goal of this procedure is to use an efficient method to gain concurrent information from multiple tissues for protein expression in a high throughput project such as the human protein atlas. This is accomplished by first selecting representative areas of formal and fixed paraffin embedded tissues. Next marked areas are punched and the selected cores are assembled into a single paraffin block, a tissue microarray.
Then sections are cut from the TMA and immunohistochemistry is performed. Finally, the protein expression pattern is analyzed and the data is published on the human protein atlas. Ultimately, results can be obtained that show protein expression in tissues and cells through tissue microarrays and immunohistochemistry.
The main advantage of this technique over existing methods such as performing immunohistochemistry from single tissue paren blocks is that valuable biological material can be saved and the experiments are more reproducible. Another advantage of this technique is that you can cut in ENT costs as well as in laboratory processing time. Demonstrating this procedure will be Ian call Bay, a lab technician from my laboratory For obtaining a standardized length of the tissue cores.
Mark the stylet. It is recommended to leave 2.5 to 3.0 millimeter margins on each side of the paraffin block to avoid cracking the paraffin to mount the proper punches. Loosen the hex socket screws that hold the punch in place and position the punch so that the groove in the punch hub is firmly placed against the metal rod in the plastic V block.
Fasten the screws, making sure the edge of the metal clip is horizontal. Insert the recipient punch on the left and the donor punch on the right. Use the XY adjustment knobs and their digital micrometer readouts to move the punches along the X and Y axi axes.
To reset the machine, press the zero a BS button to toggle between inches and millimeters, press the inch millimeter button. When using a one millimeter punch for a nine by eight template, the space between the holes should be two millimeters as measured between the centers of the holes to prevent pushing the recipient punch too far down and damaging the punch, set the maximum depth for pushing the punch by placing an empty cassette into the holder and using the depth stop screw at the upper left corner of the Z guide, set the bottom position of the recipient punch to one millimeter above the bottom of the cassette. After selecting and marking the relevant area on a tissue section and designing a template for tissue microarray production, put the recipient block in the holder and use the smallest screwdriver to fasten the screws.
Avoid over tightening the screws to prevent the paraffin from breaking loose from the cassette. Push the recipient punch downward approximately five millimeters into the recipient block, and use the handle on the punch to rotate the punch back and forth. Once, relieve the downward pushing pressure slowly so that the springs can move the punch up.
Use the style it to empty the punch and discard the paraffin core. Then move the turret to switch to the donor punch. Next, place the donor block bridge with the donor block over the recipient block holder.
Use a corresponding hematin and eosin stain slide where the region of interest has been marked to locate the tissue that is to be sampled Manually hold the donor block in position with the left hand and push the punch with the right hand. Do not push on the stylet. The depth stop does not block the punch motion at the proper position for the donor block, so it is important to stop pushing when the second mark is visible on the stylet.
Rotate the handle on the punch once slowly. Release the downward pressure while still holding the donor block on the donor block bridge. Remove the bridge and the donor block.
Make sure that the donor punch is aligned with the hole that was made earlier. Adjust the punch position using the set screws. If the donor punch and the hole in the recipient block are not aligned, carefully push the punch downward until its tip reaches the top of the hole in the recipient block.
While holding this position, use the stylet to empty the tissue core into the hole. The core surface should be in alignment with the block surface. Move the turret back to the recipient punch.
Move to the next position with the XY adjustment knobs and repeat the process again by punching a sample from the recipient block until the array is complete. To remove the recipient block, loosen the screws in the holder. Put the block face down on a glass slide place on an appropriate rack, and bake the block at 42 degrees Celsius for 40 minutes.
Remove the slide and block from the oven and flatten the surface of the block by gently stroking the glass slide. Place the block with the glass slide on a cooling plate for 10 minutes. Remove the glass slide from the block.
The block is now finished and ready for sectioning. After sectioning staining and digitalizing the tissue microarray with an antibody to a protein of interest. Search for the protein in the human protein atlas by going to the website www.proteinatlas.org.
Enter the name of a protein of interest ensemble id, uni prote accession number or the HGNC name, for example, insulin. Click on the protein of interest and scroll down to normal tissue and organ summary. The overview shows protein expression in the pancreas.
Click on the tab. More tissue data. The normal tissues view sorted by organ will come into view.
Click on pancreas to view the IHC results for three antibodies directed towards the same protein. Click on an image to view a high resolution image of pancreas In this figure, examples of the quality of tissue microarrays are shown here, a straight and properly aligned array with sufficient space between the rows and columns and the edge of the paraffin. Here, some of the cores are too close to the edge, probably resulting in problems while sectioning.
This tissue microarray was baked for too long and collapsed without the correct template. Here, a properly cut section with straight and aligned columns is stained with hemat, toin, and eosin. A bad or dirty blade could result in splitting as shown here or compressed sections like this.
Germinal center cells in lymph nodes show an optimally titrated antibody resulting in a specific cytoplasmic staining without background as seen here. Negative or weak staining is found when the concentration of primary antibody is too low or if the target protein is not present in the immuno stained tissues. This tissue section is overs stained from too high of a concentration of primary antibody.
The dark color results in difficulties in determining the subcellular location due to spillover, cross reactivity and false positivity. After watching this video, you should have a good understanding of how to produce a tissue marker array and how to use it when analyzing protein expression performing immunohistochemistry. You should also have a good understanding how to browse the human protein atlas for protein expression in human tissues and cells.
