TITLE: Immunohistochemistry
AUTHORS: Kristin Anderson, Sunni Farley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps used to perform immunohistochemistry on murine and human tumor samples.</div><div class = "text-block">In brief: Study animals underwent full necropsies, including gross examination of all organs for macroscopic disease. Harvested tissues were fixed in 10% neutral buffered formalin (10:1 formalin:tissue volume ratio) for at least 48 hours, embedded in paraffin, sectioned (4mm) and used for various immunohistochemical and histochemical assays. Sections were stained with hematoxylin and eosin (H&E) or primary antibodies for markers of interest. Slides were baked for 30 minutes at 60°C and deparaffinized on the Leica Bond-Max Automated Immunostainer. Following antigen retrieval (see table below), slides were blocked for 5 minutes at room temperature with Leica Bond Peroxide Block, then for 20 minutes (25°C) with 10% normal goat serum (Jackson ImmunoResearch, cat: 005-000-121) in TBS or TCT buffer (Casein based TBS with Tween20). After primary antibody staining for 30 minutes or 60 minutes at 25°C, Leica Bond Polymer was applied for 8 minutes (25°C). Leica Bond Mixed Refine (DAB, cat: D59800) detection was performed at 2x for 10 minutes (25°C), and a Leica hematoxylin counterstain (Biocare, cat: NM-HEM-M) was added for 3-4 minutes. Slides were cleared with xylene and mounted with synthetic resin mounting medium and a 1.5 cover slip. </div><div class = "text-block">Slides were scanned in brightfield with 20x objective using a Nanozoomer Digital Pathology slide scanner (Hamamatsu; Bridgewater, New Jersey) or the Aperio ScanScope AT (Leica Biosystems). The digital images were imported into Visiopharm software (Hoersholm, Denmark) or Aperio eSlide Manager (Leica Biosystems) and analyzed after identifying regions of interest and excluding normal tissue.</div><div class = "text-block">Equipment needed:</div><div class = "text-block">Sakura Tissue-Tek VIP6 AI Vacuum Infiltration Processor</div><div class = "text-block">Sakura DRS2000 H&E Stainer (and staining racks/arms)</div><div class = "text-block">Leica Bond Rx Autostainer</div></div>

[STEPS]
?. [Grossing and specimen processing]
Gross one specimen at a time at the Mopec grossing station.Carefully verify the cassette information to the specimen ID on the specimen container.All contaminated biohazard bags must be disposed of in the approved biohazard containers.Load the cassette holding rack into Sakura Tissue-Tek VIP6 AI.Process samples on Program 8 (see program table below).
?. [Grossing and Specemin Processing]
Program number: 8Full Name: 8 Hour ProcessingInitial   Step     Reagent           Temp (0C)         Time            3          75% Ethanol     --                      40:00 min            4          95% Ethanol     --                      40:00 min            5          95% Ethanol     --                      40:00 min            6          100% Ethanol   --                      40:00 min            7          100% Ethanol   --                      40:00 min            8          Clear-Rite         --                      40:00 min            9          Clear-Rite         --                      40:00 min            10        Clear-Rite         --                      40:00 min            11        Paraffin             60C                  40:00 min            12        Paraffin             60C                  40:00 min            13        Paraffin             60C                  40:00 min            14        Paraffin             60C                  40:00 min
?. [Grossing and specimen processing]
1. Ensure the Tissue-Tek TEC embedding station paraffin reservoir is full of paraffin before the processor has completed and the cassettes need to be removed from the processor.  If the reservoir needs to be filled, transfer molten paraffin from the TBS Liquid Paraffin Dispenser.2. Remove samples from the Sakura Tissue-Tek VIP6 AI and transfer them to the warming reservoir in the Tissue-Tek TEC embedding station.3. Samples must be embedded immediately, or RNA degradation will occur.  4. Remove one cassette from the cassette holding reservoir at a time for embedding.5. Using a square base mold, fill with molten paraffin from the dispenser, remove the cassette lid, carefully remove the specimen with forceps and orient the tissue on edge making sure it is flush and secure on the bottom of the mold.6. Place the cassette on the top of the mold and top off with molten paraffin; move on to the cooling plate.7.Trim FFPE blocks using the Wax Trimmer.
?. [Cutting FFPE slides]
1. Turn the water bath on and fill with distilled water; heating element should be set at 37°C.2. Bring Histocool block out from freezer, fill up with distilled water. Wait 10-15 minutes before pre-cooling FFPE blocks.3. Prior to cutting: Match each FFPE block with the corresponding RFS form and accompanying slides to verify the identifiers match.4. Place the blocks (tissue side down) on the HistoCool block; pre-cooling paraffin blocks is necessary to obtain quality sections.5. Allow the blocks to soak on the ice for approximately 20 minutes (depending on fixation).6. Face each block to obtain a full tissue section.7. Replace microtome blades as necessary, in order to avoid cutting artifact.8. Place ribbons onto water bath, picking up sections on corresponding slides.9. Remove the FFPE blocks from the HistoCool block and pat dry all excess water from the blocks before placing in the plastic bag to return to investigator.10. In any of the cassettes do not have tissue, or if there were possible problems during embedding that prevent sectioning, annotate the specimen ID on the RFS requisition form.11. Place slides in slide-drying rack, allow slides to air-dry overnight.12. Dry paraffin sections at 60°C 1 hour.13. Remove the sections from the oven and allow cooling at room temperature.
?. [H&E staining]
1. Load slides into the black H&E slide staining rack(s) and attach to a plastic staining arm.2. Open the Sakura DRS2000 by gently pushing on the bottom door and place the staining arm (with the attached rack(s)) in the S1/S2 loading station; close the door.3. Press the F1 [START] button on the console, the Method at the top of the console should read F FPE H&E.4. If the correct staining method is not displayed, Press the F2 [METHOD] and the up and down buttons until the correct staining protocol is highlighted.5. Press the F1 [START] button to start the staining process.6. When staining program finishes (indicated by stainer beeping), press the F1 [REMOVE] button, open the door, carefully remove the E1/E2 Xylene bucket (Program End), and carry to the coverslipping area.7. Transfer the slide racks to one of the Xylene buckets at the coverslipping area8. Place the Xylene bucket back into the Program End station, close the stainer door, and press the F1 [CONFIRM] button.
?. [H&E staining]
Program Name: FFPE H&EStep     Reagent           Time1          Xylene             3:00 min2          Xylene             3:00 min3          Xylene             3:00 min4          100% ETOH       2:00 min5          100% ETOH       2:00 min6          95% ETOH         2:00 min7          95% ETOH         2:00 min8          80% Ethanol     2:00 min9          Water (Rinse)   1:00 min10        Hematoxylin    10:00 min11        Water (Rinse)   1:00 min12        Clarifier             0:10 min13        Water (Rinse)   2:00 min14        Bluing Solution 0:30 min15        Water (Rinse)   4:00 min16        95% Ethanol     1:00 min17        Eosin Y            Phloxine          1:30 min18        95% Ethanol     0:30 min19        95% Ethanol     1:00 min20        95% Ethanol     1:00 min21        100% Ethanol   2:00 min22        100% Ethanol   2:00 min23        100% Ethanol   2:00 min24        Xylene             3:00 min25        Xylene             3:00 min26        Xylene             Program End
?. [H&E staining]
1. Coverslip the slides using Cytoseal XYL and a VWR 24x40 coverglass.2. Wipe the back of the slide and drain the sides thoroughly to remove excess mounting media/xylene, then place on a cardboard palette to dry overnight.3. Fill out the H&E QC sheet and place the slides and the sheet in the QC bin for the Director/Ops Manager’s QC approval.
?. [IHC STAINING PROTOCOL – Mouse Polymer DAB]
ABCD1Step
Reagent
Dispense Amount
Time
21
*(Leica) Peroxide Block
150uL
5min 0sec
32
*(Leica) Bond Wash
150 uL
0sec
4&nbsp;
*(Leica) Bond Wash
150 uL
0sec
53
*(Leica) Bond Wash
150 uL
0sec
64
TCT Buffer
150 uL
10min0sec
75
*(Leica) Bond Wash
150 uL
0sec
8&nbsp;
*(Leica) Bond Wash
150 uL
0sec
95
Primary/Isotype
150 uL
0sec
10&nbsp;
Pimary/Isotype
150uL
60min0sec
116
*(Leica) Bond Wash
150 uL
0sec
127
*(Leica) Bond Wash
150 uL
0sec
138
*(Leica) Bond Wash
Open
0sec
149
*(Leica) Bond Wash
150 uL
0sec
15&nbsp;
*(Leica) Bond Wash
150 uL
20min0sec
16&nbsp;
*(Leica) Bond Wash
150 uL
2min 0sec
17&nbsp;
*(Leica) Bond Wash
150 uL
2min 0sec
18&nbsp;
*(Leica) Bond Wash
150 uL
2min 0sec
1910
Leica PowerVision anti-Mouse IgG HRP
150 uL
0sec
2011
Leica PowerVision anti-Mouse IgG HRP
150 uL
12min 0sec
2112
*(Leica) Bond Wash
150 uL
2min 0sec
2213
*(Leica) Bond Wash
Open
5min 0sec
2314
*(Leica) Bond Wash
Open
0sec
2415
*(Leica) Bond Wash
Open
2min 0sec
2516
*(Leica) Bond Wash
150 uL
0sec
2617
Deionized Water
150 uL
0sec
2718
*(Leica) Mixed DAB Refine
150 uL
1min 0sec
2819
*(Leica) Mixed DAB Refine
150 uL
9min 0sec
2920
Deionized Water
150 uL
0sec
3021
Deionized Water
Open
0sec
3122
Deionized Water
150 uL
0sec
3223
*(Leica) Hematoxylin
150 uL
0sec
3324
*(Leica) Hematoxylin
150 uL
4min 0sec
3425
Deionized Water
150 uL
0sec
3526
*(Leica) Bond Wash
Open
0sec
3627
Deionized Water
150 uL
0sec
ABCD1Step
Reagent
Dispense Amount
Time
21
*(Leica) Peroxide Block
150uL
5min 0sec
32
*(Leica) Bond Wash
150 uL
0sec
4&nbsp;
*(Leica) Bond Wash
150 uL
0sec
53
*(Leica) Bond Wash
150 uL
0sec
64
TCT Buffer
150 uL
10min0sec
75
*(Leica) Bond Wash
150 uL
0sec
8&nbsp;
*(Leica) Bond Wash
150 uL
0sec
95
Primary/Isotype
150 uL
0sec
10&nbsp;
Pimary/Isotype
150uL
60min0sec
116
*(Leica) Bond Wash
150 uL
0sec
127
*(Leica) Bond Wash
150 uL
0sec
138
*(Leica) Bond Wash
Open
0sec
149
*(Leica) Bond Wash
150 uL
0sec
15&nbsp;
*(Leica) Bond Wash
150 uL
20min0sec
16&nbsp;
*(Leica) Bond Wash
150 uL
2min 0sec
17&nbsp;
*(Leica) Bond Wash
150 uL
2min 0sec
18&nbsp;
*(Leica) Bond Wash
150 uL
2min 0sec
1910
Leica PowerVision anti-Mouse IgG HRP
150 uL
0sec
2011
Leica PowerVision anti-Mouse IgG HRP
150 uL
12min 0sec
2112
*(Leica) Bond Wash
150 uL
2min 0sec
2213
*(Leica) Bond Wash
Open
5min 0sec
2314
*(Leica) Bond Wash
Open
0sec
2415
*(Leica) Bond Wash
Open
2min 0sec
2516
*(Leica) Bond Wash
150 uL
0sec
2617
Deionized Water
150 uL
0sec
2718
*(Leica) Mixed DAB Refine
150 uL
1min 0sec
2819
*(Leica) Mixed DAB Refine
150 uL
9min 0sec
2920
Deionized Water
150 uL
0sec
3021
Deionized Water
Open
0sec
3122
Deionized Water
150 uL
0sec
3223
*(Leica) Hematoxylin
150 uL
0sec
3324
*(Leica) Hematoxylin
150 uL
4min 0sec
3425
Deionized Water
150 uL
0sec
3526
*(Leica) Bond Wash
Open
0sec
3627
Deionized Water
150 uL
0sec
?. [IHC STAINING PROTOCOL – Mouse Polymer DAB]
Dehydrate and clear the slides through the IHC dehydration line:3 changes of 95% Ethanol; 1 minute each3 changes of 100% Ethanol; 1 minute each5 changes of Xylene; 1 minute eachCoverslip the slides using Cytoseal XYL and a VWR 24x40 (or x50) coverglass.Wipe the back of the slide and drain the sides thoroughly to remove excess mounting media/xylene, then place on a cardboard palette to dry overnight.