For immunohistochemistry, formalin-fixed paraffin-embedded tissues were sliced into 4-mm-thick sections. Slides were deparafinized with xylene and heated for 15 min in citrate buffer (pH 6.0) using microwave. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol and then non-specific binding was blocked with 10% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) or mouse on mouse kit (VECTOR). The slides were incubated with primary antibodies and then incubated with appropriate secondary antibodies. Reacted antibodies were detected using ABC Elite kit (VECTOR) and diaminobenzidine (DAB) (VECTOR). Immunostaining of cultured cells was performed as described previously5 (link). Fluorescent images were obtained using TCS-SPE (Leica) and FLUOVIEW FV1200 (Olympus) confocal microscope systems.
Mouse on mouse kit
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
The Mouse-on-mouse kit is a laboratory product designed for the detection of mouse antigens in mouse tissue samples. It provides a simple and effective method for immunohistochemical staining without the interference of endogenous mouse immunoglobulins.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 sted 3x confocal microscope, by Leica (3 mentions)
Prolong gold, by Thermo Fisher Scientific (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Mab360, by Fujifilm (2 mentions)
Entellan, by Merck Group (2 mentions)
Appropriately labeled secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Imaris stitcher, by Oxford Instruments (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Tcs spe, by Leica (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
Fluoview fv1200, by Olympus (1 mentions)
Griffonia simplicifolia isolectin b4, by Thermo Fisher Scientific (1 mentions)
Fluorescence conjugated cross adsorbed secondary antibody, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Vectabond, by Vector Laboratories (1 mentions)
33 protocols using mouse on mouse kit
1
Immunohistochemistry of Formalin-Fixed Tissues
2
Retinal Vasculature and Microglial Analysis
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OIR- mice at P17 were euthanized and perfused successively with PBS and 4% PFA, and the intact retinas were collected. Retinas were blocked and permeabilized in PBS containing 10% goat serum and 1% Triton-X-100 (Sigma-Aldrich) for 30 min. Endogenous Fc receptors and IgG were blocked with rat anti-mouse CD16/CD32 (Mouse BD BlockTM, 1:50, BD Biosciences, 553142) and the blocking reagent provided in the mouse-on-mouse kit (Vector Laboratories, Cat. No. FMK-2201, Burlingame, CA, USA), respectively. Retinas were then incubated with primary antibodies against mouse Adora2a (1:100, Millipore, 05-717), rabbit IBa1 (1:400, Sakura Finetek, 019-19741, Torrance, CA), and Alexa488- or Alexa-594 labeled Griffonia simplicifolia isolectin B4 (1:200, Invitrogen, 121411 and 121413, Carlsbad, CA, USA) overnight at 4 °C, followed by incubation with fluorescence-conjugated cross-adsorbed secondary antibody (1:500, Molecular Probes, Life Technologies, A-21131, Carlsbad, CA,USA) for 1 hour, and then counterstained with DAPI (Invitrogen). Retinas were flat mounted on microscope slides in mounting medium (Vectashield; Vector Laboratories) and examined by confocal microscopy (Zeiss 780; Carl Zeiss, Jena, Germany). Areas of vaso-obliteration and vitreoretinal neovascular tufts were quantified using Adobe Photoshop CS 5 software.
3
Immunocytochemical Analysis of Retinal Cell Receptors
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Retinal cells were isolated as previously described [33 (link)]. Briefly, mouse retinas were prepared in sterile 0.9% saline. Approximately 200 mg of retinal tissue was incubated with saline containing 0.4 mg/ml papain for 30 min at 37 °C. The tissues were triturated with a siliconized pipette and dissociated single cells were centrifuged at 500 × g for 10 min. The cells were fixed in 4% paraformaldehyde for 10 min, centrifuged, and washed with saline. The cells were then placed onto a Superfrost Plus slide (Fisher Scientific) treated with Vectabond (Vector Labs) using a Cytospin4 (Thermoscientific) and stored at −80 °C until used for immunocytochemistry. For immunocytochemistry, the cells were permeabilized in methanol, incubated with 1% SDS in 0.01M PBS, and washed in 1X PBS. The slides were then incubated with 1% sodium borohydride, blocked using the mouse on mouse kit (Vector Labs), and colabeled with GS and the BMP receptor antibodies (BMPR1A, BMPRIB, and activin-like kinase receptor 2 [ALK2]). Following overnight incubation with the primary antibodies, the slides were incubated with the appropriate secondary antibodies with biotin streptavidin amplification used for the receptor antibodies. The slides were then washed, counterstained with Hoechst solution, and mounted with Aqua polymount.
4
Immunodetection of Viral dsRNA in Spinal Cord
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Slides containing 30 μm cross-sections of 4% PFA fixed spinal cord of mock and MHV-3-infected mice were washed with 1% bovine serum albumin. Then, endogenous peroxidase blockade was performed with 0.3% hydrogen peroxide and unspecific reactions were blocked with protein block (Abcam ab64226). The slides were washed with TBS-Triton X-100 0.1%, treated with fc-blocking reagents (Mouse-on-mouse kit; Vector) and incubated overnight with the primary antibody anti-dsRNA (1:50, Merck MABE1134, Clone rJ2). On the following day, the slides were washed with TBS-Triton X-100 0.1%, and the secondary antibody (biotinylated goat anti-mouse IgG, Mouse-on-mouse kit) was added for 10 min, followed by an avidin-peroxidase procedure. The reaction was revealed with DAB chromogenic solution.
5
Immunohistochemical Analysis of Brain Tissue
6
Immunohistochemical staining protocol
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Samples were deparaffinized and rehydrated through gradually descending series (100%, 95%, and 70%) of ethanol. Antigen retrieval (DAKO) was performed using a pressure cooker. After cooling on ice for 1 hour, the sections were incubated in 3% H2O2 for 30 minutes to block endogenous peroxidase activity. The sections were washed twice with PBS and incubated with Protein Block Serum Free (DAKO, X0909) for 1–2 hours at room temperature to reduce nonspecific signals. The Mouse on Mouse Kit (Vector Laboratories, BMK-2202) was then used according to the manufacturer’s instructions. The sections were incubated with primary Abs overnight at 4°C. After washing 3 times with PBS, sections were incubated with HRP-conjugated secondary Abs (DAKO P0447) for 15 minutes at room temperature. DAB (DAKO) was used for the development of Abs, and Mayer’s Hematoxylin (DAKO) was used for counterstaining. Each experiment was performed with identical durations for DAB development.
7
Immunofluorescence Staining of Muscle Samples
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Immunofluorescence and histological staining were performed as previously described 55 . Gastrocnemius (GC) muscle samples were flash‐frozen in liquid nitrogen, 2‐methylbutane solution. Frozen sections (10‐μm) were fixed with 4% paraformaldehyde for 10 minutes. Sections were washed with phosphate‐buffered saline and permeabilized with 0.3% Triton X‐100 for 5 minutes, and then blocked with Seablock (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. Cells or frozen sections were incubated at 4°C overnight with a primary antibody as follows: rabbit antidystrophin (1:200, Abcam, Cambridge, MA), antidystrophin‐PE (1:300, SC Biotechnology, Santa Cruz, CA), rabbit anti‐laminin 1 antibody (1:100, Abcam, Cambridge, MA), mouse anti‐Pax7 (1:100, DSHB, Iowa), or mouse antiembryonic myosin heavy chain (1:50, eMyHC, DSHB, IA). A Mouse on Mouse kit (Vector Laboratories, Burlingame, CA) was used per conditions according to the manufacturer's protocol. Secondary antibodies used were donkey anti‐rabbit or anti‐mouse Alexa 488‐ and Alexa 594‐conjugated IgG (Invitrogen and Jackson ImmunoResearch, West Grove, PA, respectively) per the manufacturer's instructions. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1:1,000, Invitrogen). Image acquisition was performed with a Leica Microsystems Inc. (Buffalo Grove, IL) camera at ×4–40 magnification.
8
Immunohistochemistry of Brain Tissue
9
Cardiomyocyte Identification and Necrosis
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Following OGD, cells were stained with propidium iodide (PI), washed extensively, fixed with 4% paraformaldehyde, and counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) to identify nuclei. Necrotic cells were identified as having PI-stained nuclei. Additionally, NMCMs were co-stained with a mouse monoclonal anti-cardiac troponin T antibody (cTnT, MA5-12960, Fisher Scientific, Inc., Waltham, MA, U.S.A.) using a Mouse on Mouse Kit (Vector Laboratories, Burlington, ON, Canada), biotinylated secondary, and tertiary labeling with streptavidin conjugated with AlexaFluor 488 (Life Technologies, Burlington, ON, Canada) to identify cardiomyocytes.
10
Immunofluorescence Protocol with Tyramide Amplification
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Immunofluorescence experiments were performed according to previously published procedures with minor modifications (Sreepathi and Ferraguti, 2012 (link)). Primary antibodies (Table 1 ) were prepared in 2% NGS and 0.1%Triton X-100 in TBS (TBS-T). After incubation with primary and secondary antibodies (Table 2 ), the fluorescence signal for VIP was enhanced using a tyramide signal amplification kit (TSA Fluorescein System, PerkinElmer). For the enhancement, sections were first incubated in streptavidin-HRP (diluted 1:500 in 2% NGS in TBS-T) for 30 min at room temperature and then, after two washes in TBS, in a fluorophore tyramide solution for 6 min. Sections were then mounted onto gelatin-coated slides and coverslipped with Vectashield (Vector Laboratories). When mouse monoclonal antibodies were used, sections were pretreated with a Mouse-on-Mouse kit (Vector Laboratories) to reduce endogenous mouse Ig staining.
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