Tissues were fixed in 10% buffered formalin and embedded in paraffin and sections were taken at 4 μm. Sections were deparaffinised in xylene and rehydrated using a series of graded ethanol washes. Staining was performed using the Bond RX Autostainer (Leica). Heat-Induced Epitope Retrieval was performed in an EDTA buffer pH 9 (Leica, AR9640) for 40 min at 93 °C. Slides were quenched in Peroxide Block (Leica, DS9800). Primary antibodies were used as follows: COL12A1 (1:150) Abcam ab121304; aSMA (1:150) ab5694 Abcam; pMLC2 (1:100) Cell Signalling Technologies #3671. Staining used the Leica Bond Polymer Refine Detection Kit (Leica, DS9800) as per the manufacturer’s instructions. Skeletal muscle was used as a positive control tissue for optimisation of COL12A1 staining. Staining was visualised using Diaminobenzidine (DAB). H&E staining and counterstaining were performed on the Leica ST5010 Autostainer XL (Leica). Quantification of the DAB area was performed in ImageJ (v2.3.501). Whole stained tissue sections were imaged using an Aperio slide scanner.
Bond rx autostainer
Manufactured by Leica camera
Sourced in United States, Germany
The BOND RX autostainer is a laboratory equipment designed for automated immunohistochemistry and in situ hybridization staining. It provides consistent and reliable results by automating the staining process. The core function of the BOND RX is to perform the various steps involved in sample preparation, reagent application, and staining, ensuring standardized and reproducible staining outcomes.
Automatically generated - may contain errors
Lab products found in correlation
Rnascope 2.5 ls reagent kit brown, by Advanced Cell Diagnostics (3 mentions)
Bond polymer refine detection kit, by Leica camera (3 mentions)
Basescope, by Advanced Cell Diagnostics (2 mentions)
Er2 solution, by Leica camera (2 mentions)
Leica bond wash solution, by Leica camera (2 mentions)
Antibody diluent, by Abcam (2 mentions)
Dapi solution, by Akoya Biosciences (2 mentions)
Opal multiplex automation kit reagents, by Akoya Biosciences (2 mentions)
Hematoxylin, by Leica camera (2 mentions)
Peroxide block, by Leica camera (1 mentions)
Leica bond polymer refine detection kit, by Leica camera (1 mentions)
Leica st5010 autostainer xl, by Leica camera (1 mentions)
Vectra automated quantitative pathology imaging system, by PerkinElmer (1 mentions)
A0098, by Agilent Technologies (1 mentions)
Mf48000, by Thermo Fisher Scientific (1 mentions)
Sc542, by Agilent Technologies (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Antibody diluent, by Leica camera (1 mentions)
Troma 3, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti human p selectin, by R&D Systems (1 mentions)
34 protocols using bond rx autostainer
1
Immunohistochemical Analysis of Extracellular Matrix
2
Histological and Immunohistochemical Analysis of Mouse Pancreatic Tissues
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Organs were fixed in a 10% formalin solution for 24-48hrs and stored in 70% ethanol. Formalin-fixed mouse pancreata and tumor tissue were embedded in paraffin, sectioned to obtain 4μm thick sections, and stained with hematoxylin and eosin (H&E) for histological analysis.
For immunohistochemistry, tissue sections were stained using a Bond Rx autostainer (Leica) [32 (link)]. Amylase (1:400, CST 3796), rat antibody-cytokeratin 19 (TROMA-III) (1:150, Developmental Studies Hybridoma Bank, University of Iowa), and CD3 (DAKO, A0452) were diluted in antibody diluent (Leica). Images were taken using the VECTRA® Automated Quantitative Pathology Imaging system (PerkinElmer, Hopkinton, MA, USA). For estrogen receptor alpha (ER) (1:500, Santa Cruz, SC-542) and progesterone receptor (PR) (1:200, DAKO, A0098) staining, the manufacturer’s recommended protocol (Vector laboratories, VECTASTAIN ABC HRP Kit, PK-6101) was followed. For F4/80 (1:500, Invitrogen MF48000) immunofluorescence staining, deparaffinized and rehydrated slides were microwaved in sodium citrate solution. After blocking, primary antibody was incubated at 4 degree overnight and secondary antibody (Alexa Fluor 594, 1:500) for 1 hour at room temperature. Washed slides were mounted with mounting solution with DAPI.
For immunohistochemistry, tissue sections were stained using a Bond Rx autostainer (Leica) [32 (link)]. Amylase (1:400, CST 3796), rat antibody-cytokeratin 19 (TROMA-III) (1:150, Developmental Studies Hybridoma Bank, University of Iowa), and CD3 (DAKO, A0452) were diluted in antibody diluent (Leica). Images were taken using the VECTRA® Automated Quantitative Pathology Imaging system (PerkinElmer, Hopkinton, MA, USA). For estrogen receptor alpha (ER) (1:500, Santa Cruz, SC-542) and progesterone receptor (PR) (1:200, DAKO, A0098) staining, the manufacturer’s recommended protocol (Vector laboratories, VECTASTAIN ABC HRP Kit, PK-6101) was followed. For F4/80 (1:500, Invitrogen MF48000) immunofluorescence staining, deparaffinized and rehydrated slides were microwaved in sodium citrate solution. After blocking, primary antibody was incubated at 4 degree overnight and secondary antibody (Alexa Fluor 594, 1:500) for 1 hour at room temperature. Washed slides were mounted with mounting solution with DAPI.
3
Cryosectioning and Immunostaining of Fibrin 3D-Bioinks
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Fibrin 3D-bioink samples embedded in OCT were cryosectioned into 5-μm-thick sections by a cryostat (Leica). Sections were stained by hematoxylin and eosin (H&E) and for the relevant markers using antibodies. Immunostaining was performed using BOND RX autostainer (Leica). Briefly, slides were incubated with goat serum (10% goat serum in PBS + 0.02% Tween 20 + 0.02% gelatin) for 30 min to block nonspecific binding sites. Slides were then added with rabbit anti-mouse/human GFAP, rabbit anti-mouse IBA1, and mouse anti-human P-selectin (1:20 dilution; R&D Systems). Following 1 hour of incubation, slides were incubated with the following secondary antibodies for an additional 1 hour: goat anti-rabbit Alexa Fluor 488 for GFAP and IBA1 and goat anti-mouse Alexa-594 for human P-selectin and muse P-selectin. They were then washed and treated with ProLong Gold mounting with DAPI or Hoechst 33342 before being covered with coverslips. Images were recorded using the EVOS FL Auto cell imaging system.
4
Quantitative Analysis of Murine Pancreatic Tumor Immune Infiltrates
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Thirty-seven tissue samples from murine pancreatic tumors were fixed in 10% neutral buffered formalin and received for histopathology. Tissues were processed routinely, embedded in paraffin, sectioned at 4 mm, and stained with H&E. Five additional unstained sections from each tumor were submitted for immunohistochemical staining with rabbit monoclonal antibodies for pan-macrophage marker IBA-1 (Abcam, clone EPR16589, 1:8,000), and T cell markers: CD3 (Abcam clone SP7, 1:500), CD8 (Cell Signaling Technologies, clone D4W27, 1:400), CD4 (Cell Signaling Technologies, clone D7DZZ, 1:400), and FOXP3 (Cell Signaling Technologies, clone D608R, 1:200) using a Leica Bond Rx autostainer. Whole slide imaging was performed using Leica Biosystems Aperio AT2 digital slide scanner. Digital slides were viewed and representative images were captured at 20 × magnification using Aperio ImageScope Software v12.4.3.7001. Each biomarker was quantified via digital image analysis using tuned algorithms in Leica Image Analysis Software in eSlide manager Spectrum Version 12.4.3.5008. Quantitative data was exported into an excel file and analyzed in GraphPad Prism Software Version 9 via one-way ANOVA; p value < 0.05 was considered significant.
5
Stem Cell Marker Expression in Colorectal Cancer
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ISH staining was performed on 4 μm-thick FFPE sections of six LGCA and six HGCA tissue samples and their patient-matched NC tissue samples. This was carried out on the Leica BOND™ RX Auto-stainer using probes for OCT4 (NM_002701), SOX2 (NM_003106), NANOG (NM_024865), KLF4 (NM_004235) and c-MYC (NM_002467), using the ViewRNA eZ Detection Kit to detect the presence of mRNA (Affymetrix, Santa Clara, CA, USA). Positive controls were human seminoma for OCT4, NANOG and KLF4, normal skin for SOX2, and normal colon tissue for c-MYC. To determine specificity of the probes, negative controls were run using a probe for Bacillus (NM_L38424).
6
In situ Hybridization of Stem Cell Markers
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In situ hybridisation detection for Lgr5 (312178), Olfm4 (311838), Axin2 (400338), Bcl9 (529268) and Bcl9l (466698) mRNA (All Advanced Cell Diagnostics) was performed using RNAscope 2.5 LS (Brown) Detection Kit (Advanced Cell Diagnostics) on a Bond Rx autostainer (Leica) strictly according to the manufacturer’s instructions. Basescope (Advanced Cell Diagnostics) ApcEx14 #701641 (detects wild-type Apc exon 14) was used according to the manufacturer’s instructions.
7
Immunohistochemical Staining of Tonsil Tissue
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In a Leica BOND RX autostainer, deparaffinize tonsil tissue that is placed on a charged coverslip, wash (100% ethanol ×3, Leica BOND Wash solution ×3), antigen retrieval (Leica ER2 solution, EDTA pH 9.0) for 20 min at 95 °C, wash 5× with Leica BOND Wash solution, Leica 3-4% hydrogen peroxide peroxidase blocking solution (5 min), wash 3× with Leica BOND Wash solution (0 min per wash), staining with CD3e (1;200 dilution, clone, Abcam, cat. no. ab52959) primary antibody diluted in Abcam antibody diluent (Abcam, cat. no. ab64211) for 30 min, wash 3× with Leica BOND Wash solution (2 min per wash), incubate with goat anti-rabbit secondary antibody HRP conjugate ready-to-use solution (Leica, cat. no. PV6119) (8 min), wash 3× with Leica BOND Wash solution (2 min per wash), incubation with 500 nM tyramide-barcode and 500 nM tyramide-barcode in TE buffer pH 8.0 (15 min), incubation in DAPI solution (Akoya, cat. no. FP1490) diluted 3 drops per 1 mL of TBS pH 8.0 for 5 min, wash 1× with Leica BOND Wash solution (2 min per wash), collect images.
8
Lung Tissue Processing for Histology
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Antibiotic-treated mice were infected with a nonlethal dose of 1 × 108 CFU by intranasal instillation, and at 18 h postinfection, the lungs were harvested and placed into 10% neutral buffered formalin (VWR) for 72 h. Tissue was processed by the NYU Experimental Pathology Research Laboratory. Tissues were processed into formalin-fixed paraffin-embedded (FFPE) tissue blocks using a Leica Peloris automated tissue processor. Five-micrometer paraffin sections were stained with hematoxylin and eosin (H&E) on a Leica BondRX autostainer, according to the manufacturer’s instructions. Slides were imaged on a Hamamatsu Nanozoomer whole-slide scanner.
9
Hepatic Protein Expression Analysis
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Hepatic expression and distribution of PDGFRα and viral protein 3 (VP3) were measured by immunohistochemistry. FFPE livers sectioned at 5-μm thickness were collected on Leica Microsystems Plus Slides (Leica Biosystems, Buffalo Grove, IL). Immunostaining was performed using a Leica BOND RX Autostainer (Supplementary Methods section in Supplementary Data ).
10
Dual IF IHC Staining Protocol for Protein Localization
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Protein localization was performed on three LGCA and three HGCA and their patient-matched normal colon samples by dual IF IHC staining, carried out on the Leica BOND™ RX Auto-stainer. Secondary antibodies used were Vectafluor Excel goat anti-mouse 488 (ready-to-use; cat#DK2488, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor donkey anti-rabbit 594 (1:500; cat#ab150076, Life Technologies, Carlsbad, CA, USA). All stained slides were mounted as previously described [29 (link)]. Negative controls were performed using matched isotype controls for both mouse (ready-to-use; cat#IR750, Dako, Copenhagen, Denmark) and rabbit (ready-to-use; cat#IR600, Dako).
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