TITLE: Automated Multi-plex Immunofluorescence with TSA for CD4, CD8, FOXP3, CD21, PD1 and CD68 in Follicular Lymphoma
AUTHORS: Anna Maria Tsakiroglou, Catharine West, Sue Astley, Kim Linton, Martin Fergie, Richard Byers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Observing immune cell infiltration and spatial organisaion in the tumour microenvironment of follicular lymphoma is important for the development of novel biomarkers for prognosis and treatment selection. However the identification of multiple immune cell sub-populations is limited in routine single-plex immunohistochemical staining. </div><div class = "text-block">Our goal was to develop and validate an automated protocol for multiplex immunofluoresence using tyramide signal amplification, the Opal™ 7 color Kit (Akoya Biosciences) and the Ultra Discovery (Roche) autostainer, which would enable concurrent visualisation on the same tissue sections of:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>T follicular helper cells (CD4</span><span style = "vertical-align:super;">+</span><span style = "vertical-align:super;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>cytotoxic T cells (CD8</span><span style = "vertical-align:super;">+</span><span style = "vertical-align:super;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Tregs (CD4</span><span style = "vertical-align:super;">+</span><span>FOXP3</span><span style = "vertical-align:super;">+</span><span style = "vertical-align:super;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Macrophages (CD68</span><span style = "vertical-align:super;">+</span><span style = "vertical-align:super;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>PD1</span><span style = "vertical-align:super;">+</span><span> lymphocytes</span></li><li style = "counter-reset:ol0;list-style-type:disc;">DAPI nuclear counterstain</li></ul></div><div class = "text-block">We aimed in addition to observe the spatial arrangement of these cells in reference to the follicle areas, therefore the CD21 marker, which is expressed in mature B cells and follicular dendtritic cells located in the follicles was added in the panel. </div><div class = "text-block">We validated quantitatively the agreement between single-plex and multiplex assays by comparing stained area in sequential sections of a follicular lymphoma FFPE tissue micro-array (TMA).  The TMA was constructed from a follicular lymphoma cohort of 44 patients collected retrospectively from the archives of the Christie NHS Foundation Trust (Manchester), with ethical permission granted by the Central Manchester Multi-centre Research Ethical Committee (03/08/016). The protocol was automated with the DISCOVERY ULTRA IHC/ISH Roche autostainer and stained areas were quantified using the Indica Labs’ Area Quantification Fluorescence module (HALO software).</div><div class = "text-block"><span>Statistically significant linear correlations were seen between single-plex and multiplex assays (R² >0.72, p<7.1× 10</span><span style = "vertical-align:super;">-14</span><span>, see attached) for all markers.Therefore, this multiplex immunofluorescence (mIF) protocol can be used to conserve tissue and observe immune cell spatial organisation in follicular lymphoma.</span></div></div>

[STEPS]
?. [Building a spectra library]
A spectral library is initially built using positive controls of follicular lymphoma tissue to enable unmixing the 6 fluorophore spectra and DAPI in the multi-plex experiment. To build the library, the spectrum of each individual fluorophore is acquired from single-plex experiments.The single-plex experiments are run with the same parameters and positions as in the multiplex experiment; all the pre-treatment steps are kept but only one antibody and its fluorphore are applied in the same order as for the multi-plex.The order of antibodies and corresponding fluorophores:Table 1. Order of antibodies and corresponding fluorophores.  ABC1PositionAntibodyFluorophore21CD4Opal 62032CD68Opal 65043CD8Opal 54054CD21Opal 57065FOXP3Opal 52076PD1Opal 69087DAPIThe single-plex experiments are described below. The antibodies and fluorophores can be either pipetted manually on the slide by interupting the automated protocol or prepared in a dispenser and applied automatically. For the multi-plex experiment which lasts 17 hours, we suggest the use of dispensers, whereas the single-plexes can be done manually with more flexibility.The dilutions of antibodies and fluorophores can be found in Tables 2 and 3, respectively and are the same for multi-plex and single-plex experiments.
ABC1PositionAntibodyFluorophore21CD4Opal 62032CD68Opal 65043CD8Opal 54054CD21Opal 57065FOXP3Opal 52076PD1Opal 69087DAPI
?. [Building a spectra library]
Extracting the fluorophore spectraVectra 3.5 microscope is used to take snapshots of stained areas in the single-plex slides (withouth DAPI).Snapshots are taken as multispectral images (MSI). Exposure times are set automatically for the filters relevant to each fluorophore. The filters where we do not expect to see signal from a fluorophore are set to the default value (150 ms for the MSI).InForm 2.4 software (PerkinELmer) is used to build the spectral library from these snapshots.
When auto-exposing, the exposure times at 20x magnification should each be between 20-100ms for all filters. Higher exposure times would mean that the signanl is in fact mostly autofluorescence. Lower exposure times could result in bleed through between the different markers in the multi-plex experiment. If exposure times need to be increased, the fluorophores can be diluted further to lower the signal's brightness.Cells we see as positive for each fluorophore should appear only on the filters where we expect the fluorophore to emit. If these cells are seen in unexpected filters then they may be autofluorescing red blood cells instead of true positives, or the fluorophore might be too bright and should be diluted more.
When using the new spectral library to unmix the single-plex snapshots used to build it (and any other images) the signal to noise ratio, i.e. the intensity units on stained cells versus the intensity of the background, should be ideally more than 10:1, and at least 5:1.
?. [Multiplex experiment]
Multi-plex stainingThe protocol is fully automated, except for the final steps, and lasts approximately 17 hours. For this reason we suggest running overnight and carrying out the final cycle (which is manual), washing, DAPI and coverslipping in the morning.It is important that the single-plex experiments are satisfactory before the multi-plex experiments are started.Preparation before startingAll antibodies and fluorophores will be pre-diluted and added in user fillable dispensers to be applied automatically by the autostainer. The dispensers can be used for at least a week following preparation (potentially longer but we have not tested this). The following antibody dispensers are prepared by diluting the primary concentrates with the Antibody Diluent (included in the Opal kit). At least 1ml is added in each dispenser. Table 2. Primary antibody dilutions. Antibodies are diluted with the antibody diluent included in the Opal kit.  AB1AntibodyVolume Ratio2CD4Pre-diluted3CD81:4504CD681:405FOXP31:606CD211:257PD11:150Light sensitive dispensers are preferably used for the fluorophores. The fluorophores are diluted with the Tyramide Signal Amplification liquid that is included in the Opal kit.Table 2. Fluorophore dilutions. Fluorophores are diluted with the Tyramide Signal Amplification liquid included in the Opal kit.  AB1FluorophoreFluorophore Dilution (Volume ratio)2Opal 5201:753Opal 5401:1504Opal 5701:605Opal 6201:1506Opal 6501:757Opal 6901:75
AB1AntibodyVolume Ratio2CD4Pre-diluted3CD81:4504CD681:405FOXP31:606CD211:257PD11:150
The PD1 antibody should only be used withing 2 days of diluting and preparing the dispenser. Thus the steps of the final cycle (6th) when PD1 is applied are preferably done by manually pipetting freshly prepared reagent onto the slide. Therefore no dispensers are prepared for PD1 and the corresponding Opal 690 fluorophore.
AB1FluorophoreFluorophore Dilution (Volume ratio)2Opal 5201:753Opal 5401:1504Opal 5701:605Opal 6201:1506Opal 6501:757Opal 6901:75
Caution: User fillable dispensers if prepared incorectly could dispense less or more than the nominal 100μl per 'drop'. Please refer to manufacturer's instructions.
?. [Multiplex experiment]
Scanning and unmixingWhile scanning the multi-plex images all filters are auto-exposed in the Vectra microscope.After scanning multispectrally at the same resolution as the single-plexes, the markers can be unmixed in inForm 2.4 using the spectral library.
?. [Building a spectra library]
CD4/620 Single-plexDeparafinisationIn the autostainer:Warm up slide to 69°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes1st Cycle Warm up slide to 36°CAutomatically apply 'one drop' of  primary CD4 antibody (pre-diluted dispenser) and incubate for 16 minutesApply one drop of Ultramap anti-Rb HRP secondary and incubate for 16 minutesHand-pipette 100μl of the 620 diluted with TSA and incubate for 16 minutes2nd CyclePre-treatment: Warm up slide to 95°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )2nd CyclePre-treatment: Warm up slide to 95°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanningThe automated protocol that should be programmed in the Ventana Discovery Ultra autostainer can be found here:
?. [Multiplex experiment]
DeparafinisationIn the autostainer:Warm up slide to 69‎°C and incubate for 3 cycles of 8 minutes each
?. [Multiplex experiment]
Pre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes
?. [Multiplex experiment]
1st Cycle Warm up slide to 36‎°CAutomatically apply 'one drop' of  primary CD4 antibody (pre-diluted) and incubate for 16 minutesApply one drop of Ultramap anti-Rb HRP secondary and incubate for 16 minutesApply one drop of the 620 dispenser and incubate for 16 minutes
?. [Multiplex experiment]
2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )Warm up slide to 36‎°CApply one drop of CD68 dispenser and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesApply one drop of the 650 dispenser and incubate for 16 minutes
?. [Multiplex experiment]
3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36°CApply one drop of CD8 dispenser and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesApply one drop of the 540 dispenser and incubate for 16 minutes
?. [Multiplex experiment]
4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutesWarm up slide to 36‎°CApply one drop of CD21 dispenser and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 24 minutesApply one drop of the 570 dispenser and incubate for 16 minutes
?. [Multiplex experiment]
5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CApply one drop of FOXP3 dispenser and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesApply one drop of the 520 dispenser and incubate for 16 minutes
?. [Multiplex experiment]
6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand - apply (pipette) 110μl of diluted PD1 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand - apply 110μl of diluted 690 and incubate for 16 minutes
?. [Multiplex experiment]
WashingRemove the slides from the autostainer and wash with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).
?. [Multiplex experiment]
DAPIDilute DAPI 1:2000 with PBS, add one drop on the slidesRest in a dark slide box for 10 minutesRinse with TBST then water
?. [Multiplex experiment]
CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
CD68/650 Single-plexDeparafinisationIn the autostainer:Warm up slide to 69°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95°C and incubate for 16 minutes ( CC1 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted CD68 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100μm  650 diluted with TSA and incubate for 16 minutes3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
CD8/540 Single-plexDeparafinisationIn the autostainer:Warm up slide to 69 ‎°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted CD8 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100 μm of diluted 540 and incubate for 16 minutes4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted CD8 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100 μm of diluted 540 and incubate for 16 minutes4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
CD21/570 Single-plexDeparafinisationIn the autostainer:Warm up slide to 69‎°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutesWarm up slide to 36°CHand-pipette 100 μm of diluted CD21 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 24 minutesHand-pipette 100 μm of diluted 570 and incubate for 16 minutes5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutesWarm up slide to 36°CHand-pipette 100 μm of diluted CD21 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 24 minutesHand-pipette 100 μm of diluted 570 and incubate for 16 minutes5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutesWarm up slide to 36°CHand-pipette 100 μm of diluted CD21 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 24 minutesHand-pipette 100 μm of diluted 570 and incubate for 16 minutes5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
FOXP3/520 Single-plexDeparafinisationIn the autostainer:Warm up slide to 69‎°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted FOXP3 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100 μm of diluted 520 and incubate for 16 minutes6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted FOXP3 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100 μm of diluted 520 and incubate for 16 minutes6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted FOXP3 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100 μm of diluted 520 and incubate for 16 minutes6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand-pipette 100 μm of diluted FOXP3 and incubate for 60 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand-pipette 100 μm of diluted 520 and incubate for 16 minutes6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
PD1/690 Single-plexDeparafinisationIn the autostainer:Warm up slide to 69‎°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand - apply (pipette) 110μl of diluted PD1 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand - apply 110μl of diluted 690 and incubate for 16 minutes3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand - apply (pipette) 110μl of diluted PD1 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand - apply 110μl of diluted 690 and incubate for 16 minutes4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand - apply (pipette) 110μl of diluted PD1 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand - apply 110μl of diluted 690 and incubate for 16 minutes5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand - apply (pipette) 110μl of diluted PD1 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand - apply 110μl of diluted 690 and incubate for 16 minutes6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )Warm up slide to 36‎°CHand - apply (pipette) 110μl of diluted PD1 and incubate for 32 minutesApply one drop of Ultramap anti-Ms HRP secondary and incubate for 16 minutesHand - apply 110μl of diluted 690 and incubate for 16 minutesWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
DAPIIn this step the slide is run through the autostainer, without application of antibodies or fluorophores. Then it is removed, washed and DAPI is applied.DeparafinisationIn the autostainer:Warm up slide to 69‎°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).DAPIDilute DAPI 1:2000 with PBS, add one drop on the slidesLet rest in a dark slide box for 10 minutesRinse with TBST then waterCoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning
?. [Building a spectra library]
AutofluorescenceThe spectrum of autofluorescence can also be captured by running a slide through the autostainer deparafinisation and pretreatments without application of any antibodies and fluorophores, nor DAPI. DeparafinisationIn the autostainer:Warm up slide to 69‎°C and incubate for 3 cycles of 8 minutes eachPre-treatment and blockingWarm up slide to 95°C and incubate for 40 minutes ( CC1 )Apply one drop of DISCOVERY Inhibitor and incubate for 4 minutes2nd CyclePre-treatment: Warm up slide to 95‎°C and incubate for 16 minutes ( CC1 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )3rd CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )4th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )5th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CyclePre-treatment: Warm up slide to 90‎°C and incubate for 8 minutes ( CC2 )6th CycleWashingRemove the slides from the autostainer and was with EZ prep diluted 1:10 with distilled water (3 x 5 minutes).CoverslipTwo drops of prolong gold antifade reagent (without DAPI) is used per slideAfter coverslipping, let slides dry in a dark place for at least 2 hours before scanning