TITLE: mIHC staining (OHSU, Coussens&#39; lab) SOP  (TMA_TNP)
AUTHORS: Courtney Betts, Lisa Coussens, Shamilene Sivagnanam, Konjit Betre

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes multiplex immunohistochemical staining of human FFPE tissues. A staining "cycle" is defined as being one heat-mediated antigen retrieval step followed by either 1 or 2 or 3 staining "rounds". A "round" is defined as protein block, primary antibody incubation, secondary antibody incubation, chromogen development, whole slide scanning (visualization), and chromogen removal (with alcohol). If 2+ antibodies are used within a cycle a HRP inactivation step is used between rounds in order to block any chromogen development from the first round from contributing to signal in the second round. The multiple rounds in a single cycle must use antibodies produced in different species, i.e. rabbit and mouse, in order to ensure single target chromogen amplification. </div></div>

[STEPS]
?. [Bake and Deparaffinize slides ]
Incubate slides at 58-60 for 30 min. Cool for 5 minutes, then bake for another 30 mins.
?. [Bake and Deparaffinize slides ]
Deparaffinize slides:Xylene 2 x 5 min, 100% ETOH 2 x 2 min, 95% ETOH 2 x 2 min, 70% ETOH 2 x 2 min, 50% ETOH 1 x 2 min, diH20 2 x 2 min
?. [Bake and Deparaffinize slides ]
Counterstain in Hematoxylin (Dako S3301) for 1min, diH20 4-5 dips, then put in TBST.
?. [Bake and Deparaffinize slides ]
Cover section (Cover Glass Signature Series, Thermo Scientific) with TBST (0.1 M TRIS-HCl, pH 7.5, 0.15 M NaCl plus 0.05% Tween-20).
?. [Bake and Deparaffinize slides ]
Scan the slides @ 20x magnification with the Leica Aperio slide scanner. Scan according to slide scanning protocol. Of note- first time slides are scanned requires setting-up slide settings (aka presets, same scan area and focus points) that will be used to scan that slide for the entire rest of the multiplex protocol.
?. [Bake and Deparaffinize slides ]
Agitate the slides 3-5 minutes in TBST, then gently remove coverslip.
?. [Bake and Deparaffinize slides ]
Wash slides in dH20 (4-5 dips).
?. [Perform Antigen Retrieval ]
Make antigen retrieval solution: Dilute 10x citrate #HK087-5K BioGenex (diluted from 10x with milliQ H2O) to make a 1x stock. You will need 250 mls total.
?. [Perform Antigen Retrieval ]
Perform antigen retrieval (AR): Cover plastic jar and microwave at full power for 90-120 seconds until liquid is boiling. Place slides into boiling citrate and place into the steamer for 20 minutes (make sure steamer has water in the basin).
?. [Perform Antigen Retrieval ]
Allow slides to cool down at room temperature (20-30 min).
?. [Perform Antigen Retrieval ]
Wash slides in diH20 (4-5 dips) and TBST (1 x 1 min).
?. [Block endogenous peroxidase activity ]
Incubate slides with Dako dual endogenous enzyme block (Dako catalog number S2003) for 10 minutes @ RT.
?. [Block endogenous peroxidase activity ]
Wash slides in diH20 (4-5 dips) and TBST (1x 1 min).
?. [Protein blocking ]
Block section in Blocking buffer (5% normal goat serum, 2.5% BSA, 1X PBS) for 10 min @ RT (use about 100-200µl /section).
?. [Protein blocking ]
Aspirate blocking buffer (or drain off). No washing necessary.
?. [Primary Antibody Incubation ]
Apply Primary antibody (100-200 µl/section) to section (dilute in 0.5X block buffer). Incubate in humidified chamber at 30 min @RT or overnight @4°C.
?. [Primary Antibody Incubation ]
TBST wash, 3 x 2 min with agitation.
?. [Secondary Antibody (HRP conjugated) Incubation ]
Incubate slides in primary antibody-targeting secondary antibodies conjugated with HRP-polymer (e.g. HistoFine(M/R/G) Simple Stain MAX PO, Nichirei Bioscience Inc, 1-2 drops/section) @RT for 30 mins.
?. [Secondary Antibody (HRP conjugated) Incubation ]
TBST wash, 3 x 2 min with agitation.
?. [Secondary Antibody (HRP conjugated) Incubation ]
Wash slides in diH20 briefly. Do not mix TBST and AEC solution.
?. [Development of chromogen (AEC), i.e. visualization]
Prepare AEC solution according to manufacturer's recommendation  (Vector Lab, SK-4200).
?. [Development of chromogen (AEC), i.e. visualization]
Pipette AEC solution onto tissue, being sure to completely cover tissue.
?. [Development of chromogen (AEC), i.e. visualization]
Incubate 5-40 min (usually 20 min, depends on the marker) @RT.
?. [Development of chromogen (AEC), i.e. visualization]
Wash in diH20 (1 min, to de-activated AEC enzymatic reaction), then place slides in TBST.
?. [Perform whole slide scanning]
Cover section (Cover Glass Signature Series, Thermo Scientific) with TBST. Drain excess fluid, and dry bottom of slide to remove streaks.
?. [Perform whole slide scanning]
Scan the slides @ 20x magnification with the Leica Aperio slide scanner. See slide scanning protocol for details.
?. [Perform whole slide scanning]
Agitate slides in TBST for 3-5 minutes, then gently remove coverslip.
?. [Remove chromogen with alcohol ]
Put slides into diH20 briefly, 70% ETOH briefly, 100% ETOH with agitation until signal-clearence (usually 2-3 min). BE SURE to check under the microscope that all color has been removed.
?. [Remove chromogen with alcohol ]
Rehydrate slides: 70% ETOH 1 x 1 min, 30% ETOH 1 x 1min, dH20 wash 4-5 x briefly (make sure for complete elimination of EtOH), TBST 1 x 1min
?. [Remove chromogen with alcohol ]
At this stage- go to step 8 if need to start next cycle. Go to step 31 if need to start a new round (within a cycle).
?. [Inactivation of HRP on secondary antibodies ]
Incubate slides with Dako dual endogenous enzyme block (Dako catalog number S2003) for 10 minutes @ RT.
?. [Inactivation of HRP on secondary antibodies ]
Wash slides in diH20 (4-5 dips) and TBST (1x 1 min).
?. Go to step 16 in order to add next primary antibody (to complete next round).