Primary antibody diluent

Manufactured by Leica

Sourced in Germany

Primary antibody diluent is a solution used to dilute and stabilize primary antibodies for immunohistochemistry and other immunoassays. It helps maintain the activity and specificity of the primary antibodies.

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Lab products found in correlation

Citrate based solution ph 6.0, by Leica (3 mentions) Polymer refine red detection kit, by Leica (3 mentions) Micromount, by Leica (3 mentions) Bond rxm platform, by Leica (2 mentions) Hl344, by Cell Signaling Technology (2 mentions) Biosystems refine detection kit, by Leica (1 mentions) Ix81 inverted microscope, by Olympus (1 mentions) Nuance multispectral imaging system, by PerkinElmer (1 mentions) Eclipse ci upright microscope, by Nikon (1 mentions) Bond automated immunostainer, by Leica (1 mentions) Inform analysis software, by PerkinElmer (1 mentions) Ab16667, by Abcam (1 mentions) Axioscan slide scanner, by Zeiss (1 mentions) Polymer refine detection kit, by Leica (1 mentions) Confirm anti total c met sp44 rabbit monoclonal primary antibody, by Roche (1 mentions) Bond rx autostainer, by Leica (1 mentions) Ab174316, by Abcam (1 mentions) Gtx105085, by GeneTex (1 mentions) Refine detection kit, by Leica (1 mentions) Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)

10 protocols using primary antibody diluent

Dual Chromogenic IHC for Microbial Detection

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Single immunohistochemistry was performed on the Leica Bond III automated staining platform using the Leica Biosystems Refine Detection Kit (DS9800). All antibodies were diluted in Leica Primary Antibody Diluent (AR93520). The primary antibodies listed in Table 1 were used at the dilutions indicated. No antigen retrieval was used for any of the primary antibodies.
Dual chromogenic IHC Immunohistochemistry was performed sequentially on the Leica Bond III automated staining platform using the Leica Biosystems Refine Detection Kit (DS9800) and Leica Biosystems Refine Detection Kit (DS9390). All antibodies were diluted in Leica Primary Antibody Diluent (AR93520). The primary antibodies listed in Table 1 were used at the dilutions indicated. For one pair of antibodies, Candida albicans was visualized via 3, 3'-diaminobenzidine (DAB) and S. mutans was visualized via alkaline phosphatase (AP). For another antibody pair, fungal β-D glucan was visualized via DAB and gram-positive bacteria LTA was visualized via AP. For a third antibody pair, gram-negative endotoxin was visualized via DAB and gram-positive bacteria LTA was visualized via AP. For a fourth antibody pair, β-D glucan was visualized via DAB and gram-negative endotoxin was visualized via AP. The coverslips were then mounted and the slides were visualized using an Olympus IX81 inverted microscope.

Sulyanto R.M., Beall C.J., Ha K., Montesano J., Juang J., Dickson J.R., Hashmi S.B., Bradbury S., Leys E.J., Edgerton M., Ho S.P, & Griffen A.L. (2024). Fungi and bacteria occupy distinct spatial niches within carious dentin. PLoS pathogens, 20(5).

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Automated Immunohistochemistry for SARS-CoV-2 Detection

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For immunohistochemistry (IHC), 4-μm sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost Plus slides and subjected to IHC using an anti-nucleocapsid rabbit monoclonal antibody (HL344, Cell Signaling Technology, Danvers, MA). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems, Wetzlar, Germany). Following automated deparaffinization, heat-induced epitope retrieval was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody (diluted at 1:1,600 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 min), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60°C oven for 30 min and mounted with a permanent mounting medium (Micromount, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.

Hall J.S., Hofmeister E., Ip H.S., Nashold S.W., Leon A.E., Malavé C.M., Falendysz E.A., Rocke T.E., Carossino M., Balasuriya U, & Knowles S. (2023). Experimental Infection of Mexican Free-Tailed Bats (Tadarida brasiliensis) with SARS-CoV-2. mSphere, 8(1), e00263-22.

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SARS-CoV-2 Nucleocapsid Protein IHC Protocol

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For IHC, four-micron sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost Plus slides and subjected to IHC using anti-nucleocapsid rabbit polyclonal antibody (3A, developed by our laboratory) with the method previously described (80 (link)). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems). Following automated deparaffinization, heat-induced epitope retrieval (HIER) was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody [anti-SARS-CoV-2 nucleocapsid rabbit polyclonal antibody diluted at 1:5,000 in primary antibody diluent (Leica Biosystems)] for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 min), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60°C oven for 30 min and mounted with a permanent mounting medium (Micromount, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.

Carossino M., Izadmehr S., Trujillo J.D., Gaudreault N.N., Dittmar W., Morozov I., Balasuriya U.B., Cordon-Cardo C., García-Sastre A, & Richt J.A. (2024). ACE2 and TMPRSS2 distribution in the respiratory tract of different animal species and its correlation with SARS-CoV-2 tissue tropism. Microbiology Spectrum, 12(2), e03270-23.

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Immunohistochemistry for SARS-CoV-2 Nucleocapsid

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For IHC, 4 μm sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost® Plus slides and subjected to IHC using an anti-nucleocapsid rabbit monoclonal antibody (HL344, Cell Signaling Technology, Danvers, Massachusetts). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems, Wetzlar, Germany). Following automated deparaffinization, heat-induced epitope retrieval (HIER) was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody (diluted at 1:1,600 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 minutes), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60 °C oven for 30 min and mounted with a permanent mounting medium (Micromount®, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.

Hall J., Hofmeister E., Ip H., Nashold S., Leon A., Malavé C., Falendysz E., Rocke T., Carossino M., Balasuriya U, & Knowles S. (2022). Experimental infection of Mexican free-tailed bats (Tadarida brasiliensis) with SARS-CoV-2. bioRxiv.

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EGFR Expression in Glioblastoma Samples

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Tissue microarrays containing duplicates of 33 glioblastoma cases, 2 brain anaplastic astrocytoma, and 5 normal brain tissue samples were ordered from US Biomax, Inc. (Cat: GL806c). Arrays were stained with primary mouse anti-human EGFR antibody (Clone 31G7, Invitrogen) at a 1:100 dilution. Briefly, slides were baked for 30 minutes at 60°C and deparaffinized on the Leica Bond Automated Immunostainer, followed by antigen retrieval with Proteinase K for 10min at 37°C. Blocking was performed for 10min at RT using Normal Goat Serum (10% in TBS) followed by primary antibody in Leica primary antibody diluent for 30min. Secondary antibodies (Life Technologies) were incubated with TMAs for 2hr at RT and diluted 1:500 in PBS with 0.2% BSA.
TMAs were imaged using the Nuance Multispectral imaging system (Perkin Elmer) on a Nikon Eclipse Ci upright microscope at 20 ×. Images were captured every 20 nm wavelength of light from 420 nm-720 nm. Data were analyzed by InForm analysis software (Perkin Elmer) using a threshold of 0.07OD.

Ravanpay A.C., Gust J., Johnson A.J., Rolczynski L.S., Cecchini M., Chang C.A., Hoglund V.J., Mukherjee R., Vitanza N.A., Orentas R.J, & Jensen M.C. (2019). EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma. Oncotarget, 10(66), 7080-7095.

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Quantifying Pancreatic Cell Proliferation

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Pancreas samples were fixed in 4% paraformaldehyde prior to paraffin embedding (FFPE). Three-micron tissue sections were de-waxed with xylene and rehydrated in a graded ethanol series. Slides were analysed via immunohistochemistry. For this, samples were blocked with 3% hydrogen peroxide and antigen retrieval was performed by heating the sections in Citrate buffer (95 °C; pH 6.0) for 30 min. Sections were incubated with the following primary antibody: Ki67, rabbit monoclonal, Ab 16667 (abcam). Antibody was diluted 1:100 with Leica Primary Antibody Diluent (AR9352; Leica Biosystems, Nussloch, Germany) and incubated for 1 h at room temperature. Bond™ Polymer Refine Detection (Cat# 37072) was applied for visualization. Staining was performed on the automated Leica IHC Bond-RX™ platform (Leica Biosystems, Nussloch, Germany). Image generation was conducted with a Zeiss AxioScan slide scanner (Zeiss, Oberkochen, Germany). With the use of HALO® (Indica Labs, UK) as Digital Image Analysis platform, Ki-67 positive cells were identified with brown nuclear markup. Mitotic index was defined as the ratio between the numbers of Ki67 positive cells to the total number of cells.

Loeffler M., Klepac K., Baljuls A., Hamilton B., Mayer-Wrangowski S., Haebel P, & Zimmermann T. (2023). Effects of a long-acting secretin peptide analog alone and in combination with a GLP-1R agonist in a diet-induced obesity mouse model. Molecular Metabolism, 74, 101765.

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Profiling Tumor Progression in MET-driven Lung Cancer

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Patients receiving targeted therapies at Massachusetts General Hospital undergo repeat biopsy at time of progression and are enrolled in an institutional review board-approved protocol of tissue collection and research. A patient who was receiving crizotinib therapy for metastatic lung cancer with MET exon 14 skipping underwent a biopsy at time of progression. Tumor genotyping on the baseline and postprogression biopsy samples was performed with the Snapshot nextgeneration sequencing version 1 assay, which uses anchored multiplex polymerase chain reaction for the detection of single nucleotide variants and insertion/ deletions in the genomic DNA of 39 cancer-related genes. 10 Immunohistochemical testing was performed on 5-mm sections of formalin-fixed paraffin-embedded tumor tissue. Total MET staining was performed using the CONFIRM Anti-Total c-MET (SP44) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, AZ), and phospho-MET staining was performed using the Anti-MET phospho Y1349 rabbit monoclonal primary antibody (EP2367Y; ab68141) (Abcam, Cambridge, MA) diluted 1:100 in Leica Primary Antibody Diluent (Leica Biosystems Richmond, Inc., Richmond, IL). Targets were retrieved using Epitope Retrieval Solution 2 for 20 minutes, and staining was performed on the Leica BOND RX autostainer using the Polymer-Refine Detection Kit.

Heist R.S., Sequist L.V., Borger D., Gainor J.F., Arellano R.S., Le L.P., Dias-Santagata D., Clark J.W., Engelman J.A., Shaw A.T, & Iafrate A.J. (2016). Acquired Resistance to Crizotinib in NSCLC with MET Exon 14 Skipping. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 11(8).

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Immunohistochemical Analysis of Lung Fibrosis

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Normal and fibrotic lung tissue samples from the BLM-induced mouse model were prepared and Masson trichrome stained as described previously^{27 (link)}. Human IPF and healthy lung tissue was purchased at Folio bioscience (Columbus, Ohio, USA) following the guidelines of the ethics committee. For IHC, lung tissue was processed using standard procedures, embedded in paraffin, and mounted on SuperfrostUltra Plus slides. Antigen retrieval was performed in citrate based buffer, pH 6.0. Antibodies: LOX: ab174316 (Abcam, Cambridge, UK) (dilution 1:300); LOXL1: NBP1-82827, (Acris Antibodies GmbH, Germany) (dilution 1:100), LOXL2: GTX105085 (GeneTex Inc., Irvine, CA) (dilution 1:500), LOXL3: LS-C165846 (LSBio, Seattle, WA) (dilution 1:500), LOXL4: sc-48731 (SantaCruz, Inc., Dallas, TX). Antibody dilutions were carried out in primary antibody diluent (Leica, Germany).

Aumiller V., Strobel B., Romeike M., Schuler M., Stierstorfer B.E, & Kreuz S. (2017). Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis. Scientific Reports, 7, 149.

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Dual Immunofluorescence Staining of Fungal and Bacterial Markers

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Dual immunofluorescence staining was performed sequentially on the Leica Bond III automated staining platform using the Leica Biosystems Refine Detection Kit (DS9800). All antibodies were diluted in Leica Primary Antibody Diluent (AR93520). For one pair of antibodies, Candida albicans was visualized via Alexa Fluor 647 (Life Technologies; B40958) and S. mutans was visualized via Alexa Fluor 555 (Life Technologies; B40955. For another antibody pair, fungal β-D glucan was visualized via Alexa Fluor 647 (Life Technologies; B40958) and gram-positive bacteria LTA was visualized via Alexa Fluor 647 (Life Technologies; B40958). For a third antibody pair, gram-negative endotoxin was visualized via Alexa Fluor 647 (Life Technologies; B40958) and gram-positive bacteria LTA was visualized via Alexa Fluor 555 (Life Technologies; B40955). The coverslips were then mounted and visualized using an Olympus Fluoview FV1000 Confocal microscope.

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Immunohistochemical Assessment of ACE2 and TMPRSS2

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Harvested tissues were fixed in 10% phosphate-buffered formalin for 24–48 h. Following fixation, tissues were embedded in paraffin, sectioned at 5 μm, mounted on glass slides and deparaffinized prior to the staining procedures. The sections were immunostained with a BOND-MAX system (Leica Microsystems GmbH, Wetzlar, Germany) using the manufacturer’s reagents according to the protocol as described.‍^{(11 (link))} Anti-ACE2 (1:250, ab108252; Abcam, Cambridge, UK) and anti-TMPRSS2 (1:8000, ab92323; Abcam) antibodies were used after diluting with Primary Antibody Diluent (Leica). Tissue array slides (MNO341; US BIOMAX Inc., Rockville, MD) were used for the analysis of various organs in humans and as positive controls. Regarding the evaluation of ACE2 immunostaining, we classified the samples into three groups depending on the staining intensity in epithelial cells. To secure the reproducibility, two pathologists independently evaluated them, which was assessed with κ coefficient. In terms of TMPRSS2 evaluation, we classified them into three groups: strong (216–256 out of 256-level density), moderate (186–215), weak (156–185) with ImageJ software (National Institutes of Health, Bethesda, MD) (Supplemental Fig. 1*).

Sato K., Fujii K., Yamamoto N., Ichimura N., Yamaguchi S., Yamada H., Hibi H, & Toyokuni S. (2022). Association of alcohol intake and female gender with high expression of TMPRSS2 in tongue as potential risk for SARS-CoV-2 infection. Journal of Clinical Biochemistry and Nutrition, 71(2), 129-135.

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