Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

1. How to Prepare Tissues for Tissue Microarray Production (Animation 1)

1. Select relevant formalin fixed paraffin embedded material (tissue or cell samples) including corresponding hematoxylin stained tissue section.
2. Mark relevant area on the tissue section. It is recommended to have a freshly cut hematoxylin stained section corresponding to the paraffin block.
3. Design the template used for TMA production. Randomize the samples within the template to avoid artifacts caused by technical problems such as coverage of the antibody over the whole TMA section and sectioning problems. Add additional orientation markers to the template for orientation.
4. Organize the tissues according to the template.

2. Manual Tissue Microarray Production (Essential for filming)

1. To be able to obtain a standardized length of the tissue cores, mark the stylet eg 4.5 mm (Figure 2). Guidelines for spacing between sample centers:
   core size 0.6 mm – 0.8-1.0 mm
   core size 1.0 mm – 1.8-2.0 mm
   core size 1.5 mm – 2.0-3.0 mm
   core size 2.0 mm – 2.5-4.0 mm
   It is recommended to leave 2.5-3.0 mm margins on each side of the paraffin block to avoid cracking of the paraffin.
   
2. 1. Mount the selected punches to be used. Start by loosening the hex socket screws that holds the punch in place. The punch is correctly positioned when the groove in the punch hub is firmly placed against the metal rod in the plastic v-block. Fasten the screws and make sure that the edge of the metal clip is horizontal.
   2. The recipient punch (marked in green and red, specific for distributor) should be placed to the left. The donor punch (marked in green and blue, specific for distributor), which has a slightly larger diameter, should be placed to the right (Figure 3).
3. 1. Move the punches along the x- and y-axes by using the XY adjustment knobs. The right adjustment knob moves the punches along the x-axis and the left adjustment knob moves the punches along the y-axis.
      There is a digital micrometer readout on both adjustment knobs. These are activated when moving the knobs.
   2. Reset → press the ZERO/ABS button.
      Shift between inches and mm → press the IN/mm button.
      The space between the holes should be 2.0 mm (measured between the centers of the holes) when using a 1 mm punch for a 9×8 template.
4. 1. To ensure that you do not push the punch too far down into the block, hitting the cassette and destroying the punch, put a cassette without paraffin in the holder and set the bottom position of the recipient punch to 1 mm above the bottom of the cassette by using the depth stop screw at the upper left corner of the Z-guide (Figure 3).
   2. Put the recipient block in the holder and use the smallest screwdriver to fasten the screws. Do not fasten the screws to tight or the paraffin might break loose from the cassette.
   3. Push the recipient punch downward approximately 5 mm into the recipient block and use the handle in the punch to rotate the punch back and forth once (Figure 3). The hole will be 5 mm deep when pushing the punch as far down into the block as possible.
   4. Relieve the downward pushing pressure slowly, so that the springs can move the punch up. Use the stylet to empty the punch and discard the paraffin core.
5. Move the turret to switch to the donor punch (Figure 3).
6. Place the donor block bridge with the donor block over the recipient block holder (Figure 3).
7. 1. Push the donor punch into the selected area of the donor block. Use a corresponding hematoxylin and eosin stained 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 your left hand and push the punch with your right hand. Note that you should 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 that you stop pushing when the second mark is visible on the stylet.
   2. Rotate the handle on the punch once (Figure 3). Slowly release the downwards pressure while still holding the donor block on the donor block bridge.
   3. The donor tissue is preferably punched 0.5 mm shorter than the recipient core. This process will ensure an adjustable alignment of the cores on the recipient block surface, avoiding loss of valuable representative tissue.
8. Remove the bridge and the donor block. Make sure that the donor punch is aligned with the hole that was made earlier (point 4). You must adjust the punch position using the set screws if the donor punch and the hole in the recipient block are not aligned.
9. 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.
10. Move the turret back to the recipient punch.
11. Move to the next position with the XY adjustment knobs.
12. Repeat from point 4c above until the array is complete.
13. When the whole recipient block is finished, loosen the screws in the recipient block holder. Bake the block in 42 °C for 40 minutes. Put the block with the sectioning area facing down on a glass slide. Place the glass slide with the block on top of a test tube holder (or other appropriate construction) and place in the oven. After baking, remove the test tube holder from the oven and flatten the surface of the block by gently stroking the glass slide.
14. Place the block with the glass slide on a cooling plate for 10 minutes.
    To ensure representative tissue throughout the TMA a quality control on each 50^th section is performed.

3. How to Section TMAs (Animation 2)

1. Cut 4 μm thick sections by using a microtome with section transfer system (HM 355S, Microm). The system can be used with all current rotary microtomes. The system consists of a blade carrier with integrated transfer bridge, a heated water bath and a control unit. From the blade the sections glide on the wet transfer surface via a transfer bridge into the water bath.
2. The sections should be taken from the water bath immediately after they have stretched out, to avoid melting of sensitive tissue spots (eg. lipid rich tissues like breast and brain). Place the section on a superfrost glass slide. The time the sections can be left in the water depends on the type of paraffin waxed used and the water temperature. Our lab use paraffin wax from HistoLab Products AB, which have a melting point of 56-58 ° C and we recommend a water bath temperature of 37-39 °C.
3. Place the slides in a slide holder for drying in room temperature (RT) over night.
4. The slides are baked in 50 °C for 12-24 hours. Using a microtom without STS you have to transport the section from the blade to a water bath manually by using e.g. a brush.

4. Test and Titration Procedure for Immunohistochemistry (Animation 3)

Each antibody is subjected to a standardized test procedure using specially designed TMAs containing a mix of tissues and different cell types. The aim is to assess an individual and optimized immunostaining protocol for each antibody. Initially one dilution, based on antibody stock concentration of the primary antibody is tested. The outcome of this test staining is used to guide further dilutions and optimization of the protocol.

1. Deparaffinization and hydration in xylene and graded ethanol to distilled water are performed prior to immunostaining.
2. A blocking step to quench endogeneous peroxidase is performed in 0.3 % H₂O₂ in 95% ethanol for 5 minutes.
3. Heat Induced Epitope Retrieval (HIER) is performed in a retrieval buffer pH 6, using a pressure boiler (Decloaking chamber, Biocare Medical) as heat source for 4 minutes at 125 °C. After completed boiling, slides remain in the pressure boiler and are allowed to cool to 90 °C. The total processing time for HIER is approximately 45 minutes.
4. If no conclusive result is obtained in the initial test, further tests are done based on the observed staining pattern ^{5 6}. A pilot study, within the HPA high-throughput set-up, showed that the most useful antigen retrieval where HIER pH 6, but other retrieval methods such as pre-incubation with proteinase, microwave and boiling in buffer with higher or lower pH can of course be used. By changing the IHC condition eg changing antibody incubation times, antibody dilutions and Diaminobenzidine (DAB) incubation times the staining pattern can give more conclusive results.

5. Staining Program, Autostainer 480 (Thermo Fisher Scientific)

All incubations are done at RT and this protocol can also be used in as a manual setup with the same reagents.

1. Rinse in wash buffer.
2. Incubation with Ultra V block for 5 minutes.
3. Rinse in wash buffer.
4. Incubation with primary antibody for 30 minutes.
5. Rinse in wash buffer (x2).
6. Incubation with labeled horse radish peroxidase-polymer for 30 minutes.
7. Rinse in wash buffer (x2).
8. Developing in DAB solution for 10 minutes.
9. Rinse in wash buffer (x2).
10. Counterstaining in Mayers hematoxylin for 5 minutes*. When using a progressive staining method (e.g. Mayers hematoxylin) the intensity depends on time and no differentiation is required. A regressive staining method e.g. Harris hematoxylin requires differentiation to remove excess dye from tissues in order to accentuate a structure.
11. Rinse in tap water*.
12. Rinse in lithium carbonate water, diluted 1:5 from saturated solution for 1 minute*.
13. Rinse in tap water for 5 minutes*.
14. The slides are dehydrated in graded ethanol and slides are coverslipped (PERTEX, Histolab)

All reagents are applied at a volume of 300 μl per slide.
* Steps 10-14 are done in a histostaining instrument (Autostainer XL, Leica) and coverslipping is additionally done in a coverslipper system (Automated glass coverslipper CV5030, Leica).

6. How to Scan a Slide

The method of scanning slides is dependant on the digital scanner being utilized, this protocol only defines how to scan a slide using the automated scanning system Aperio XT (Aperio Technologies). In the Human Protein Atlas set-up 20x magnification for the tissue and 40x for the cells are used. Automated scanning could encounter focus problems due to heterogeneity of the tissue and the number of different tissues included in a TMA.

1. Clean the glass slide from dirt, glue and check for air bubbles between the coverslip and the section. In case of air bubbles remount the slide.
2. Put the slide in a rack and place the rack in the digital scanner. The time to scan a TMA at 20x magnification is approximately 6 minutes. A software from Aperio is used to select area of interest. Focus point selection is made automatically within the software. Manually selection of scan area and focus points is also possible.
3. After the image has been generated it can be used for evaluation and annotation of the IHC staining. Images remain as a result file of the experiment and can be distributed to colleagues for discussions and further used for publishing the data. The images can be viewed in a freely available software from Aperio.

7. How to Search for your Favorite Protein in the Human Protein Atlas

1. Browse to www.proteinatlas.org.
2. Enter the name of your favorite protein, Ensembl id, UniProt accession number or HGNC name eg insulin.
3. Enter the summary page and scroll down to normal tissue and organ summary. The overview shows protein expression in pancreas.
4. Click on “more tissue data”. You enter the normal tissues view where you can see all tissues included sorted by organ.
5. Click on pancreas to view the IHC results for 3 antibodies directed towards the same protein. To view a high-resolution image of pancreas click on the image.
6. Additional information about protein expression in cancer tissue, cells and further validation data within the Human Protein Atlas can be found by browsing the Human Protein Atlas website.

8. Representative Results

Animation 1. How to prepare tissues for tissue microarray production. Click here to view Animation 1.

Animation 2. How to section TMAs. Click here to view Animation 2.

Animation 3. The immunohistochemistry technique is described. Click here to view Animation 3.

Figure 1. Overall scheme of the process within the Human Protein Atlas Uppsala.

Figure 2. Donor stylet marked for standardized length of tissue cores eg 4.5 mm.

Figure 3. Manual tissue microarrayer.

Figure 4. Examples of different quality of produced TMAs. (A) A straight and properly aligned TMA with sufficient space between the rows and columns as well as to the edge of the paraffin. (B) A TMA with some of the cores too close to the edge resulting in probable difficulties when sectioning. (C) A TMA that have been baked for too long resulting in a collapsed TMA without the correct template.

Figure 5. Hematoxylin and eosin stained TMAs. (A) A properly cut section with straight and aligned columns and rows. Using a bad or dirty blade could result in splitting (B) or compressed sections (C).

Figure 6. Titration of antibodies. (A) Reaction center cells in lymph node shows an optimally titrated antibody resulting in a specific cytoplasmic staining (shown in brown) without background. (B) The tissue section shows weak staining. 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 immunostained tissues. (C) The tissue section is overstained due to use of too high concentration of primary antibody. The dark color results in difficulties determining the subcellular location due to spill over, cross reactivity and false positivity.