Rodent decloaker

Manufactured by Biocare Medical

Sourced in United States

Rodent Decloaker is a laboratory equipment product designed for use in the preparation and processing of rodent tissue samples. Its core function is to facilitate the retrieval of target antigens from formalin-fixed, paraffin-embedded (FFPE) rodent tissue sections.

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Lab products found in correlation

Rodent block m, by Biocare Medical (3 mentions) Peroxidazed 1, by Biocare Medical (2 mentions) Bdb2004h, by Biocare Medical (2 mentions) 880 airyscan microscope, by Zeiss (2 mentions) Opal 7 color manual ihc kit, by PerkinElmer (2 mentions) Bloxall blocking solution, by Vector Laboratories (2 mentions) Immpress hrp goat anti rat igg, by Vector Laboratories (2 mentions) Dm6000 microscope, by Leica (1 mentions) Em ccd, by Hamamatsu Photonics (1 mentions) Rmr622g, by Biocare Medical (1 mentions) Rodent blocker, by Biocare Medical (1 mentions) Avidin biotin blocking kit, by BioGenex (1 mentions) Bx43 light microscope, by Olympus (1 mentions) Axiovert 200 microscope, by Zeiss (1 mentions) Axiocam mrm camera, by Zeiss (1 mentions) Alexa fluor 488 and 594, by Thermo Fisher Scientific (1 mentions) Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Donkey serum, by Jackson ImmunoResearch (1 mentions) Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)

Spelling variants of this product

Decloaker (30 citations) Rodent Decloaker solution (4 citations) Rodent decloaker buffer (2 citations)

13 protocols using rodent decloaker

Quantitative Immunofluorescence Analysis of Pancreatic Islet Cells

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Slides were dewaxed prior to heat induced antigen retrieval with Biocare Rodent Decloaker at 100 °C for 15 min. After cooling for 30 min the slides were blocked for 1 h with goat serum block. Slides were incubated overnight at 4 °C with 1:400 dilutions of both Invitrogen guinea pig anti-insulin and Cell Signaling’s rabbit anti-glucagon antibodies. After 17 h, the slides were washed in TBST prior to incubation with 1:300 dilutions of Invitrogen’s goat anti-guinea pig Alexa 488 and Invitrogen’s goat anti-rabbit Alexa 594 secondary antibodies for 1 h at RT. The slides were then washed in TBST prior to counterstaining with Hoechst.
After mounting and allowing the slides to cure for about 1–2 h, the slides were imaged on a Leica DM6000 microscope. Images were collected using a Hamamatsu EM-CCD (512 × 512 pixel) camera and were exported as single channel TIFF images for analysis in CellProfiler software. A CellProfiler pipeline was developed to identify insulin-positive beta cells as well as glucagon-positive alpha-cells based on green or red staining intensity, respectively. The area and integrated intensity of each cell type was collected and used to determine the average intensity of green (insulin) or red (glucagon) signal as well as the ratio of glucagon expressing vs insulin expressing cell area.

Hegde V., Na H.N., Dubuisson O., Burke S.J., Collier J.J., Burk D., Mendoza T, & Dhurandhar N.V. (2015). An adenovirus-derived protein: A novel candidate for anti-diabetic drug development. Biochimie, 121, 140-150.

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Immunohistochemical Analysis of CD46 Expression

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The expression of CD46 was assessed by using the immunoperoxidase method. Formalin-fixed, 4-μm paraffin-embedded kidney sections were incubated for 5 min with Peroxidazed 1 (PX968, Biocare Medical, Pacheco, CA, USA) to quench endogenous peroxidase, after antigen retrieval in Rodent Decloaker (RD913, Biocare Medical). After blocking for 10 min with Rodent Block M (RBM961G, Biocare Medical), sections were incubated with antibody anti-CD46 (1:200; PA5-95,788, ThermoFisher) followed by rabbit HPR-Polymer (RMR622G, Biocare Medical) for 30 min. The staining was visualized by the addition of the betazoid 3,3′diaminobenzidine chromogen kit solutions (BDB2004H, Biocare Medical). Finally, slides were counterstained with Mayer hematoxylin and observed through light microscopy (ApoTome). Negative controls were obtained by omitting the primary antibody on adjacent sections.

Perico L., Morigi M., Pezzotta A., Locatelli M., Imberti B., Corna D., Cerullo D., Benigni A, & Remuzzi G. (2023). SARS-CoV-2 spike protein induces lung endothelial cell dysfunction and thrombo-inflammation depending on the C3a/C3a receptor signalling. Scientific Reports, 13, 11392.

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Lung Immune Cell Profiling Protocol

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Lungs were harvested and fixed in 10% formalin for 24 hours. Fixed lungs were embedded in paraffin and 8-μM thick sections were prepared for staining (VitroVivo Biotech). Slides were stained using the Opal 7-Color Manual IHC Kit (PerkinElmer) per the manufacturer’s instructions. Briefly, slides were deparaffinized and rehydrated in a series of xylene and ethanol gradients, microwaved in Rodent Decloaker (Biocare Medical) and then cooled. Slides were washed with water and then TBST (TBS + 0.05% Tween-20) followed by blocking with Bloxall Blocking Solution (Vector Laboratories). Staining with primary and secondary antibodies and OPAL fluorophores were performed per the manufacturer’s instructions. The following antibodies were used: CD4 (Invitrogen, clone 4SM95; 1:100), CD8 (Invitrogen, clone 4SM16; 1:100), ImmPRESS HRP Goat anti-rat IgG (Vector Laboratories; 1:4). All images were collected on a Zeiss 880/Airyscan Microscope at the CCR Microscopy Core Facility using the 20X and 40X objectives. For quantitation, the 20X images were used to manually count the number of CD4 and CD8 cells and calculate nuclear area using color thresholding in ImageJ (NIH).

Kaczanowska S., Beury D.W., Gopalan V., Tycko A.K., Qin H., Clements M.E., Drake J., Nwanze C., Murgai M., Rae Z., Ju W., Alexander K.A., Kline J., Contreras C.F., Wessel K.M., Patel S., Hannenhalli S., Kelly M, & Kaplan R.N. (2021). Genetically Engineered Myeloid Cells Rebalance the Core Immune Suppression Program in Metastasis. Cell, 184(8), 2033-2052.e21.

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Immunohistochemical Staining of CD3+ T Cells in Mouse Brain

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Paraffin embedded sections of mouse brain were postfixed in neutral buffered formalin followed by antigen retrieval with Rodent Decloaker (Biocare Medical, CA, USA). Sections were blocked with avidin and biotin blocks for 20 min (Avidin Biotin Blocking Kit, Biogenex Laboratories) followed by the Rodent Blocker (Bio Care Medical, Richmond, CA, USA) and Fc receptor blocker (Innovex Biosciences) for 30 minutes at RT.
Sections were incubated with rabbit CD3 (Abcam) antibodies overnight at 4°C following secondary goat anti-rabbit antibodies (Biocare Medical, Richmond, CA, USA) for 30 min at RT. Sections were next developed with streptavidin-labeled peroxidase and Turbo DAB (Innovex Biosciences, Richmond, CA, USA) for 2–5 minutes and counterstained with hematoxylin.

Bauer D.F., Pereboeva L., Gillespie G.Y., Cloud G.A., Elzafarany O., Langford C., Markert J.M, & Jr. L.S. (2016). Effect of HSV-IL12 Loaded Tumor Cell-Based Vaccination in a Mouse Model of High-Grade Neuroblastoma. Journal of Immunology Research, 2016, 2568125.

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Immunohistochemical Analysis of Tissue

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Stomachs were fixed in 10% formalin, and paraffin embedded^{76 (link)}. Sections (5 μm) deparaffinized, treated (99 °C, 18 min) for antigen retrieval (rodent decloaker, Biocare Medical) were blocked 1 h in 1% donkey serum (Jackson Labs), 1% BSA (Sigma-Aldrich) and 0.3% Triton-X100. Primary antibodies (overnight, 4 °C) were followed by horseradish peroxidase (Jackson Immuno Research Labs) or fluorescently labeled secondary antibodies (Alexa Fluor 488 and 594; Invitrogen). Supplementary Table 1 lists primary antibodies and dilutions. Fluorescence used a Zeiss Axiovert 200 microscope with Axiocam MRM camera and Axiovision software. Bright-field images used a 2.0 HT NanoZoomer (Hamamatsu) whole slide scanner or an Olympus BX43 light microscope.

Jacome-Sosa M., Miao Z.F., Peche V.S., Morris E.F., Narendran R., Pietka K.M., Samovski D., Lo H.Y., Pietka T., Varro A., Love-Gregory L., Goldenring J.R., Kuda O., Gamazon E.R., Mills J.C, & Abumrad N.A. (2021). CD36 maintains the gastric mucosa and associates with gastric disease. Communications Biology, 4, 1247.

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Quantifying Macrophage Marker F4/80 in Tissue

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After deparaffinization and rehydration, paraffin sections were incubated with rodent decloaker (Biocare Medical, #RD913M) for 30 min at 95°C for antigen retrieval and then washed with TBS. To block nonspecific binding, sections were incubated with 10% goat serum in 5% BSA/TBS for 1 h at RT in a humid chamber under gentle agitation. Following the removal of the block buffer, sections were labeled with primary antibody against F4/80 (1:100, Santa Cruz, #sc‐377009) overnight at 4°C, washed with TBS, and then detected with Alexa Fluor 488 goat anti‐rat secondary antibody (1:200, Abcam, ab150157) in 5% BSA/TBS for 30 min at RT in a humid chamber under gentle agitation in the dark. Sections were then washed with TBS and counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1ug/ml). Sections were washed with water and mounted with a glycerol based mounting media. Representative fluorescent images were taken at ×20 using Leica DM2500 fluorescent microscope.

McDonald S.J., VanderVeen B.N., Bullard B.M., Cardaci T.D., Madero S.S., Chatzistamou I., Fan D, & Murphy E.A. (2022). Surgical wounding enhances pro‐tumor macrophage responses and accelerates tumor growth and lung metastasis in a triple negative breast cancer mouse model. Physiological Reports, 10(21), e15497.

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Quantitative Multiplex Immunohistochemistry of Tumor Immune Microenvironment

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Tumors grown in C57BL/6 and C3H mice were harvested upon reaching 12 mm in diameter and were fixed in zinc-based fixative for 24 hours at room temperature as previously described (16 (link)). Tissue was then processed by preparing 4 µm thick tissue sections, incubation of slides at 37°C and deparaffinization. The protocol included blocking with goat serum (Vector) prior to staining and dilution of primary antibodies in Renaissance Background reducing Diluent (Biocare medical). Tissue sections were boiled in Rodent Decloaker (Biocare Medical) for antigen retrieval, except prior to CD8 staining where pH=9 buffer (Perkin Elmer) was used. Primary antibodies were anti-CD3 (SP7, Abcam, Cambridge, UK), anti-CD8α (4SM15, eBioscience), anti-FoxP3 (FJK-16s, Invitrogen, Carlsbad, CA), anti-PD-L1 (D5V3B, Cell Signaling Technology, Danvers, MA), and DAPI (Perkin Elmer). Opal 7-Color Automation IHC Kit was used (690 for CD3, 570 for PD-L1, 620 for CD8α, and 520 for FoxP3). Slides were scanned with Vectra Polaris (Perkin Elmer, Waltham, MA) and analyzed using QuPath 0.2.0-m4 (17 (link)).

Sharon S., Duhen T., Bambina S., Baird J., Leidner R., Bell B., Casap N., Crittenden M., Vasudevan S., Jubran M., Kravchenko-Balasha N, & Gough M. (2021). Explant Modeling of the Immune Environment of Head and Neck Cancer. Frontiers in Oncology, 11, 611365.

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Immunohistochemical Analysis of Tissue Samples

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Immunostaining and Congo red histochemistry were performed on 5 μm paraffin sections. Antigens were retrieved in 25 mM citrate buffer (pH 7.2) or Rodent Decloaker (Biocare Medical, USA) using a microwave oven. Sections were blocked with M.O.M.™ IgG block (Vector Labs, USA) or Rodent Block M (Biocare Medical). Primary antibody (Additional file 1: Table 1) incubation was carried out overnight at ~ 4 °C followed by incubation with secondary antibody for 30–60 min at room temperature. ABC™ complex and NOVA™ red reagents (Vector Labs) were used to visualize the immunosignals. In other settings, MM AP-Polymer kit (mouse antibody on mouse tissues) or MACH3™ Rabbit-Probe Alk Phos Polymer kits were employed along with Vulcan Fast Red Chromogen kit 2 (Biocare Medical). For double immunostaining using fluorescent secondary antibodies, primary antibodies were incubated overnight, simultaneously or stepwise at 4 °C, followed by incubation with the appropriate secondary antibodies (Alexa Fluor, fluorescent secondary antibodies against mouse and rabbit, Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) was used for counterstaining of nuclei. Sulfated alcian blue staining was performed according to Lendrum et al. [28 (link)].

Zhang X., O’Callaghan P., Li H., Tan Y., Zhang G., Barash U., Wang X., Lannfelt L., Vlodavsky I., Lindahl U, & Li J.P. (2021). Heparanase overexpression impedes perivascular clearance of amyloid-β from murine brain: relevance to Alzheimer’s disease. Acta Neuropathologica Communications, 9, 84.

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Immunohistochemical Staining of FFPE Mouse Tissue

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FFPE mouse tissue sections were deparaffinized and rehydrated in gradient ethanol solutions. Antigens were retrieved in Rodent Decloaker (BioCare Medical, RD913M) and microwaved twice in an EZ Retriever System V3 (BioGenex) at 95°C for 5 minutes. Tissues were blocked in PeroxAbolish (BioCare Medical, PXA969M) for 30 minutes, Rodent Block M (BioCare Medical, RBM961L) for 30 minutes, and 5% BSA in PBS for 30 minutes. Tissues were incubated with primary antibody as indicated overnight at 4°C. VisUCyte HRP Polymer IgG (R&D Systems, VC001-025 for mouse, VC003-025 for rabbit) was applied for 30 minutes at room temperature followed by DAB chromogenesis (BioCare Medical, BDB2004L). Tissues were counterstained with CAT hematoxylin (Thermo Fisher Scientific, CATHE-M) for 20 seconds. The slides were then dehydrated through gradient ethanol solutions and 2 passes of xylene and sealed with Permount (Thermo Fisher Scientific, SP15-100).

Lu Z., Mao W., Yang H., Santiago-O’Farrill J.M., Rask P.J., Mondal J., Chen H., Ivan C., Liu X., Liu C.G., Xi Y., Masuda K., Carrami E.M., Chen M., Tang Y., Pang L., Lakomy D.S., Calin G.A., Liang H., Ahmed A.A., Vankayalapati H, & Bast RC J.r. (2022). SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers. The Journal of Clinical Investigation, 132(11), e146471.

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Immunostaining of Kidney Sections

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ED-1 and CD8 stainings were performed using the immunoperoxidase method. Duboscq-Brazil-fixed, 3 μm paraffin-embedded kidney sections were incubated for 5 min with Peroxidazed 1 (PX968, Biocare Medical, Pacheco, CA, USA) to quench endogenous peroxidase, after antigen retrieval in Rodent Decloaker (RD913, Biocare Medical). After blocking for 10 min with Rodent Block R (RBR962, Biocare Medical), sections were incubated with mouse monoclonal antibody to monocyte/macrophage ED-1 surface antigen (1:100; MAB1435 Merck Millipore, Burlington, MA, USA) or mouse anti-rat-CD8 (1:50; 550298 DB Biosciences) followed by Rat HPR-Polymer (BRR4016, Biocare Medical) for 30 min. The stainings were visualized by the addition of the betazoid 3,3′diaminobenzidine chromogen kit solutions (BDB2004H, Biocare Medical). Finally, slides were counterstained with Mayer hematoxylin and observed through light microscopy (ApoTome, Axio Imager Z2, Zeiss, Oberkochen, Germany). Negative controls were obtained by omitting the primary antibody on adjacent sections. ED-1-positive monocytes/macrophages and CD8+ T cells within glomeruli were counted in an average of 25 glomeruli and expressed as the average number of cells per glomerulus.

Cerullo D., Rottoli D., Corna D., Abbate M., Benigni A., Remuzzi G, & Zoja C. (2022). Add-On Cyclic Angiotensin-(1-7) with Cyclophosphamide Arrests Progressive Kidney Disease in Rats with ANCA Associated Glomerulonephritis. Cells, 11(15), 2434.

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