Bond dewax solution

Slides were deparaffinized using the Leica Bond Dewax solution by soaking in a Coplin staining jar for three minutes. Slides were washed 3 times in 100% ethanol, then either air dried, or continued through H&E staining with the following protocol: rehydrated in distilled water for three minutes; immersed in hematoxylin solution (Leica hematoxylin 560 diluted 1:6 in distilled water) for three minutes; washed three times in distilled water; immersed briefly in 0.1x PBS; washed three more times in distilled water; soaked in 70% Ethanol for two minutes; immersed in Alcoholic Eosin Y with Phloxine (Sigma HT110332) for three minutes; washed in 100% ethanol three times then air dried.

A validated FLRT3 mAb (Santa Cruz Biotechnology, clone A-3, catalog no. sc-514482) was used in the IHC procedure developed by Discovery Life Sciences (DLS; Newtown, PA), and outlined in Table 2 to stain FFPE human tissues from DLS tissue bank, and stained by DLS. Tissue sections were dewaxed using the Leica dewax protocol on the BOND III platform with Leica’s BOND Dewax solution. Antigen retrieval was performed after tissue sections were dewaxed using the Leica BOND III platform. For tissue pretreatment, slides were heated to 100°C for 20 min in high-pH (tris-EDTA, pH 9) Leica BOND ER2 solution. Detection was optimized with inclusion of proteinase K (1:40 dilution) enzyme to further expose the epitopes for binding during tissue pretreatment. The primary Ab was incubated for 1 hour on the Leica BOND III platform, and detection was performed using reagents from BOND Polymer Refine Detection kit from Leica for visualization of mouse primary Abs. Various concentrations of Abs were tested using Reagent Manufacturing Buffer (RMB) Ab diluent with goat serum.

Samples were fixed in formalin and embedded in paraffin; 4 μm-thick sections were stained with H&E. The frequency of GCs was calculated by manually counting the number of GCs on spleen sections and dividing this value by the total spleen section area, in a blinded manner (four mice per genotype). The ratio MZ/lymphoid follicle area was esteemed as previously described (26 (link)). Image acquisition was performed using Leica DMD108 Digital Microimaging Device and Software (Leica Microsystems, Germany). Pictures were acquired using 4x, 10x, 20x and 40x objectives at RT. IHC analysis was performed using an automated platform (Bond-maX; Leica, Newcastle Upon Tyne, UK). Tissue sections were treated with the Bond Dewax Solution (Leica) at 72°C. The CPS/EDTA or Heat/EDTA Ag retrieval methods were used, according to the manufacturer’s instructions. Abs: polyclonal BCL6 (Santa Cruz). AICDA transcript (ID: 11628) was detected using RNAscope 2·5 HD Detection Reagent-BROWN (Advanced Cell Diagnostic) in accordance with the manufacturer’s protocol. Quantitative analyses of Aicda mRNA in situ hybridization signals were performed by calculating the average percentage of positive signals in five nonoverlapping fields at high-power magnification (X400) using the Positive Pixel Count v9 ImageScope software, Leica Biosystems.

Formalin fixed, paraffin embedded tissue sections were deparaffinized using Bond Dewax Solution (Leica) after which Epitopes were retrieved using Bond Epitope Retrieval Solution 1 (Leica). The sections were stained for EV-A71 antigen (10F0, abcam), PSGL1 (bs-0561R, Bioss) or SCARB2 (NB400-129, Novus). In addition, haematoxylin and eosin staining was performed to visualize inflammation.

Formalin-fixed, paraffin-embedded tumors were sectioned at 5 microns, baked at 60°C for 30 minutes, soaked in Bond Dewax Solution (Leica), and rehydrated. Deparaffinization and staining of all slides was performed on the Leica BOND RX Autostainer (Leica). Before being used in combination, the specificity and optimal dilution of each antibody was individually determined with chromogenic immunohistochemistry (DAB) using slides from normal tonsil and head and neck carcinoma, consistent with best practice guidelines (46 (link)). Heat-induced epitope retrieval was performed by heating to 95°C in BOND epitope retrieval solutions ER1 or ER2 (Leica). Tyramide signal amplification (TSA) Opal technology was used for immunofluorescence staining. After individual primary antibody optimization, primary and secondary antibody and opal pairings were optimized for minimum background and desired signal amplification in monoplex immunofluorescence using head and neck carcinoma sections. Supplemental Table 3 lists the primary and secondary antibodies used and the Opal details. Slides were coverslipped using the Leica CV5030 automated coverslipper after staining. Whole-slide images were obtained at ×40 magnification using 7-color, whole-slide unmixing filters on a Vectra Polaris. All paired pre- and post-treatment tumors were stained and scanned concurrently.

Following standard euthanasia, grafted tissue was harvested and fixed in 10% formaldehyde (Thermo Fisher Scientific, Waltham, MA), and then tissue was embedded in paraffin. Formalin fixed paraffin sections were cut at 4 µm, placed on charged slides and dried at 60°C for 1 h. Slides were cooled to room temperature and added to the Leica Bond RX, where they were deparaffinized with Bond Dewax Solution (Leica, Allendale, NJ) and rinsed in water. Bond Epitope Retrieval Solution 2 (Leica, Allendale, NJ) was used for target retrieval for 30 min. Slides were blocked using peroxide block from a Bond Polymer Refine Detection kit (Leica, Allendale, NJ) for 5 min. Slides were incubated with CD4 Antibody (Abcam, Cambridge, United Kingdom) at 1/1000 or CD8 (Abcam, Cambridge, United Kingdom) at 1/1000 or FOXP3 (Boster Biological Technology, Pleasanton, CA) at 1/50 for 20 min followed by Rabbit Envision (Agilent Technologies, Santa Clara, CA) for 30 min. Diaminobenzidine from the Bond Polymer Refine Detection kit (Leica, Allendale, NJ) was applied for 10 min for visualization. Slides were counterstained with haematoxylin from the Bond Polymer Refine Detection kit (Leica, Allendale, NJ) for 8 min then placed into water. After removing slides from the Bond they were dehydrated, cleared and cover‐slipped.