Paraffin-embedded tissue sections were cut at 10-μm-thick. Immunohistochemical (IHC) began with microwave antigen retrieval for 6 min (power 8) using Trilogy buffer (Cell Marque; Rocklin, CA) for CD68 and Declere buffer (Cell Marque; Rocklin, CA) for IBA1. Sections were then placed in 3% H2O2 in methanol for 30 min. Following washes in distilled water, sections were blocked in 5% goat serum at room temperature for 1 h. Sections were incubated in primary antibodies IBA1 (rabbit polyclonal, 1:1,000 IHC, Wako); CD68 (clone KP1) (1:50 IHC, Dako) overnight at 4°C. A biotinylated secondary antibody (Vector Laboratories) was amplified using avidin-biotin substrate (ABC solution, Vector Laboratories catalog no. PK-6100), followed by color development in Nova Red (Vector Laboratories). Immunofluorescence (IF) staining was done following microwave antigen retrieval for 6 min (power 8) using Declere buffer (Cell Marque; Rocklin, CA) for primary antibodies to: IBA1 (rabbit polyclonal, 1:250 IF, Wako); and PHF-1 (1:500 IHC and IF, a kind gift from Dr Peter Davies, Bronx, NY), and visualized using appropriate secondary antibody conjugated to an Alexafluor probe (1:200, Lifetechnologies) applied for 1 h. A 0.1% solution of Sudan Black was used to reduce autofluorescence. Slides were coverslipped using Vectashield mounting medium with DAPI (Vector Labs, Burlingame, CA).
Trilogy buffer
Manufactured by Cell Marque
Trilogy buffer is a versatile, multi-purpose buffer solution designed for use in various laboratory applications. It serves as a general-purpose buffer for maintaining pH and ionic conditions in a wide range of experiments and procedures.
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Lab products found in correlation
Dfc365fx, by Leica (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Novared, by Vector Laboratories (1 mentions)
Declere buffer, by Cell Marque (1 mentions)
Cd68 clone kp1, by Agilent Technologies (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Gram staining kit, by Merck Group (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fluoromount g with dapi, by Thermo Fisher Scientific (1 mentions)
Image j, by Keyence (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Aperio epathology, by Leica (1 mentions)
Goat serum, by Merck Group (1 mentions)
Imagescope software, by Leica (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Vectra 3.0 pathology imaging system microscope, by PerkinElmer (1 mentions)
Halo image analysis software, by Indica Labs (1 mentions)
12 protocols using trilogy buffer
1
Immunohistochemical and Immunofluorescence Analysis
2
Immunohistochemical Analysis of MMP2 in Irradiated Mice
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Mice were anesthetized 48 hours after the last irradiation at week 16. Formalin-fixed, paraffin-embedded sections were obtained from the lower dorsum of mice, oriented perpendicularly to the wrinkles. After deparaffinization using Trilogy buffer (Cell Marque), slides were washed 3 times with PBS and blocked with 10% normal goat serum diluted in PBS with 0.05% Tween 20 (PBST) for 1 hour. Samples were then incubated with primary antibody diluted in PBST (anti-MMP2, 1:200) overnight at 4°C. Following 3 washes with PBST on an orbital shaker, samples were incubated with a secondary antibody (goat anti-mouse-A488; diluted 1:1000 in PBST) for 1 hour at room temperature on an orbital shaker, protected from light. After 3 more washes with PBST, samples were stained with DAPI (diluted 1:1000 in PBS) for 2 minutes. Samples were quickly washed 3 times in PBS, and glass cover slips were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific). After 48 hours of drying while protected from light, slides were imaged using a fluorescence microscope at 200× original magnification (Leica, DFC365FX).
3
Immunofluorescence Staining of Skin Samples
4
Mammary Gland Histology and Protein Analysis
5
IL-36α Immunofluorescence in Mouse Tissue
6
Multiplex Immunohistochemistry Analysis of Tumor Microenvironment
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Two tissue microarrays containing T/L/N samples of 52 patients were stained with Opal Multiplex Immunohistochemistry Detection Kit (Perkin‐Elmer) and images were acquired using a Vectra 3.0 Pathology Imaging System Microscope (Perkin‐Elmer). Slides were deparaffinized and rehydrated and antigen retrieved using Trilogy buffer (CellMarque) by autoclaving for 15 min. Slides were treated with 3% H2O2 for 15 min, washed, and blocked using 4% BSA/PBS/0.1% Triton X‐100 (all from Sigma). Antibodies used were: anti‐CD8, anti‐CD4, and anti‐PD‐1. Detection dye for each antibody was: Opal570 dye (CD8), Opal520 dye (CD4), and Opal620 dye (PD‐1). DAPI was used as a nuclear counterstain. The digital images were analyzed with Halo Image Analysis software (indica labs) using Highplex FL module which allows for the simultaneous analysis of up to eight immunofluorescence‐labeled markers in any cellular compartment—nucleus, cytoplasm, and/or membrane. Cells negative for all markers are black, cells positive for individual makers are colored according to that marker color, and cells positive for three markers were calculated and marked in blue in the simulation image. Antibodies used in this experiment and the corresponding dilution ratio were listed in Table S4 (Supporting Information).
7
Immunohistochemistry of Paraffin Sections
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Tissue sections embedded in paraffin were dry-baked vertically for 30 min at 55°C in Coplin jars and then cooled to room temperature. De-paraffinizing, rehydrating, and unmasking were completed using Trilogy buffer (920P-09; Cell Marque) and a 16-min antigen retrieval in a pressure cooker. The slides were washed with 1× PBS and incubated in blocking solution (PBS/1% normal donkey serum/1% BSA/0.1% Triton X-100) for 30 min at room temperature and then incubated with primary antibodies overnight at 4°C in a humidifier chamber (See Table S1 for antibodies and dilutions). No primary controls (NPCs) did not receive primary antibody on the transcription factor channel. The sections were washed for 5 min three times with 1× PBS the following day and then incubated with secondary antibodies and DAPI (1:10,000) for 1–2 h at room temperature. The sections were washed for 5 min three times with 1× PBS and mounted on SuperFrost Plus Microscope Slides (12-550-16; Thermo Fisher Scientific). Once dry, the slides were rinsed with ddH2O followed by the addition of mounting media (Mowiol) and a coverslip.
8
Immunohistochemical Detection of Apoptosis
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The carcinoma tissues collected the groups were fixed, embedded into paraffin, and cut into 4-μm-thick sections. The sections were transferred onto cover slips. The sections were dewaxed and rehydrated in Trilogy buffer (Cell Marque, Rocklin, CA) in a pressure cooker for 15 min, blocked in 5% serum for 30 min, and then incubated with antibodies against cleaved caspase-3 at Asp175 (1: 500, #P42574, Cell Signaling, Danvers, MA) at 4°C overnight. After washing, the sections were incubated in a secondary Alexa Fluor-555-labeled goat anti-rabbit IgG (#4413, Cell Signaling) for 30 min at room temperature in the dark. Cover slips were mounted with mounting media containing DAPI to stain the nuclei. The number of cleaved caspase-3-positive cells was scored by counting 3 sets of at least 100 tumor cells each under the microscope.
9
Quantifying Tissue-Resident Lymphoid Structures
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TLS maturation was analyzed in tissue sections by 7-plex multiplex IF as previously described (Silina et al., 2018, Springer Protocols) (48 ). Briefly, tissue sections were deparaffinized, rehydrated and retrieved all in one step using the Trilogy buffer (CellMarque) for 10 min at 110°C in a pressure cooker. The following antibodies and dilutions were used for a 7-plex IF; CD21 (1:5000, clone 2G9 Leica), DC-LAMP (1:1000, clone 1010E1.01, Dendritics), CD23 (1:1000, clone SP3, Abcam), PNAd (1:5000, clone MECA-79, Biolegend), CD20 (1:5000, clone L26, Dako), CD3 (1:1000, clone SP7, ThermoScientific) and 200x magnified images were acquired by Vectra 3.0 multispectral microscope (PerkinElmer/Akoya). Area segregation was done by Inform tissue segmentation algorithm of the Inform software (Akoya).
TLS maturation stages were defined by the presence or absence of CD21+ Follicular Dendritic cells (FDC) networks and CD23+ Germinal Center (GC) cells in dense CD20+ B-cell regions. Proportions of early TLS (no FDCs, no GC), primary follicle-like (PFL) TLS (has FDCs but no GC) and secondary follicle-like (SFL) TLS were determined as fractions out of all analyzed TLS for each patient.
TLS maturation stages were defined by the presence or absence of CD21+ Follicular Dendritic cells (FDC) networks and CD23+ Germinal Center (GC) cells in dense CD20+ B-cell regions. Proportions of early TLS (no FDCs, no GC), primary follicle-like (PFL) TLS (has FDCs but no GC) and secondary follicle-like (SFL) TLS were determined as fractions out of all analyzed TLS for each patient.
10
Multicolor Fluorescent IHC of ccRCC
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Formalin-fixed paraffin embedded sections from M-5 positive and control ccRCC samples were stained using the Opal 7-Color Fluorescent IHC Kit (PerkinElmer) according to manufacturer’s protocol. Slides were deparaffinized and rehydrated and antigen retrieved using Trilogy buffer (CellMarque) by autoclaving for 15 min. Slides were treated with 3% H2O2 for 15 min, washed, and blocked using 4% BSA/PBS/0.1% Triton X-100 (all from Sigma). Primary antibodies and consecutive HRP-conjugated secondary antibodies (Table S7 ) were diluted in 1% BSA/PBS/0.1% Triton X-100 and incubated for 1 hr at room temperature. Slides were then incubated in Amplification diluent containing a tyramide-conjugated fluorophore for 10 min. Prior to the second primary antibody incubation, the slides were heated for 10 min in 10 mM citric acid, pH 6.0 at 95°C to strip the antibodies of the first staining round. The protocol was repeated from the blocking step 3 to 4 times to co-stain several markers.
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