Frozen muscles were sectioned at 8 μm thicknesses using a Leica CM1950 cryostat maintained at −20℃. The muscle sections were first stained with hematoxylin and eosin (H/E) for histological assessment of xenograft regeneration. Muscle sections were also immunostained with human‐specific spectrin (SPEC1‐CE, 1:50, Leica) and human‐specific lamin A/C (ab40567, 1:200, Abcam), while total muscle tissue was marked by 4′6‐diamidino‐2‐phenylindole (DAPI) staining (outside human‐marked tissue) or by immunostaining with fluorescently conjugated wheat germ agglutinin (WGA alexafluor‐647, W32466, 1:500, Thermo Fisher Scientific). Specifically, muscle sections were fixed in ice‐cold methanol for 10 minutes, washed in phosphate‐buffered saline (PBS), and blocked in PBS supplemented with 2% goat serum (GTX73249, GeneTex) and mouse‐on‐mouse (M.O.M.) blocking reagent (MKB‐2213, Vector Laboratories). Muscle sections were incubated with primary antibodies overnight at 4℃ and subsequently probed with Alexa Fluor secondary antibodies (Life Technologies). Finally, muscle sections were mounted with Prolong Gold Mounting Media (Life Technologies) with DAPI for nuclear staining.
Mkb 2213
Manufactured by Vector Laboratories
Sourced in United States
The MKB-2213 is a laboratory instrument designed for the detection and quantification of various biological molecules. It utilizes a specialized technique to generate reliable and reproducible data. The core function of the MKB-2213 is to provide accurate measurements for research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
X0909, by Agilent Technologies (2 mentions)
Anti arid1a, by Merck Group (2 mentions)
Prolong gold mounting media with dapi, by Thermo Fisher Scientific (2 mentions)
Anti p eef2k s366, by Cell Signaling Technology (2 mentions)
Anti cytokeratin 5, by Abcam (2 mentions)
Anti cytokeratin 5, by BioLegend (2 mentions)
Aperio scanscope fl, by Leica Biosystems (2 mentions)
Anti puromycin, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Ab40567, by Abcam (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold mounting media, by Thermo Fisher Scientific (1 mentions)
Cm1950 cryostat, by Leica camera (1 mentions)
Wga alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
S1699, by Agilent Technologies (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
S0809, by Agilent Technologies (1 mentions)
18 protocols using mkb 2213
1
Histological Assessment of Xenograft Regeneration
2
Immunofluorescence Staining Protocol
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Slides were warmed on a heating block for 15 min before deparaffinization in Histoclear (National Diagnostics, HS-200). Sections were rehydrated, and antigen retrieval was performed in 1× citrate buffer, pH 6.0 (Dako, S1699), in a pressure cooker for 15 min on high-pressure setting. Slides were cooled on ice and then blocked for 1.5 h with serum-free protein block (Dako, X0909). For mouse antibodies, an additional mouse-on-mouse block (Vector Laboratories, MKB-2213) was used for 20 min at room temperature followed by a PBS wash. Primary antibodies were then incubated overnight at 4°C. Three washes with PBS were performed, and sections were incubated for 1 h at room temperature with secondary antibodies. 1:1,000 Hoechst (Thermo Fisher Scientific, 62249; 10 mg/ml) was added to each section, followed by three washes in PBS. One drop of ProLong Gold antifade reagent was added to slides to coverslip (Thermo Fisher Scientific, P36934).
3
Immunofluorescence Staining of Paraffin Sections
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Paraffin sections were warmed on a heating block set at 65°C for 10 minutes to facilitate bonding of the tissue with the glass slide. Tissue then was deparaffinized in Histo-Clear (HS-200; National Diagnostics, Atlanta, GA) and a series of ethanol, followed by rehydration and antigen retrieval in citrate buffer (S1699; Dako) in a pressure cooker set for high pressure for 15 minutes. After antigen retrieval, slides were cooled on ice and blocked at room temperature for 1.5 hours with serum-free protein block (X0909; Dako). Mouse on mouse block (MKB-2213; Vector Laboratories, Burlingame, CA) was used for mouse antibodies following the manufacturer’s instructions. Primary antibodies were diluted in antibody diluent with background reducing components (S3022; National Diagnostics) and were incubated overnight at 4°C in a humidified chamber. After 3 washes in PBS for 5 minutes, sections were incubated with secondary antibodies diluted 1:200 in antibody diluent (S0809; Dako, Santa Clara, CA) for 1 hour at room temperature. Hoechst (1:1000 in PBS, 62249, 12.3 mg/mL; Thermo Fisher Scientific) was added to stain the nuclei for 5 minutes. Sections then were washed 3 times in PBS for 5 minutes each. Coverslips were added to sections with ProLong Gold Antifade (P36934; Thermo Fisher Scientific) and allowed to dry before imaging.
4
Immunofluorescence Staining of Muscle Tissue
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Sections were blocked in 2% bovine serum albumin (BSA) plus M.o.M. (MKB‐2213, Vector Labs) for 60 min. Sections were then incubated in primary antibodies against eMyHC (supernatant 1:20; F1.652, Developmental Studies Hybridoma Bank) and laminin (1:200; L9393, Sigma‐Aldrich) diluted in 2% BSA for 90 min, washed in PBS, and incubated in secondary antibodies against Ms IgG1 AF594 (1:200; A‐21125, Invitrogen) and Rb IgG AF488 (1:200; A‐11008, Invitrogen) diluted in PBS for 60 min. Sections were washed, incubated in DAPI (1:10,000; D1306, Invitrogen) for 15 min, washed in PBS, and cover slipped using PBS and glycerol (1:1).
5
Immunohistochemical Analysis of Intestinal Tissue
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The small intestine and colon were opened and fixed for 24 h in 4% PFA. The tissue was paraffin embedded and sectioned. RFP and β-catenin immunohistochemistry were carried out using a Bond Max autostainer (Leica), with sodium citrate, pH 6.0 (10 mM) antigen retrieval. Slides were blocked with 3% hydrogen peroxide, followed by incubation using an Avidin/Biotin Blocking Kit (Vector Laboratories). Anti-RFP (1:100, Abcam ab34771) and β-catenin (BD biosciences, 610154, 0.25 μg/mL) primary antibodies were used. For β-catenin IHC, a mouse-on-mouse blocking step was added (Vector Laboratories, MKB-2213). Secondary antibodies used were biotinylated donkey and biotinylated donkey anti-rabbit (1:250, Jackson ImmunoResearch, 711-065-152) and biotinylated rabbit anti-mouse IgG1 (1:500, Abcam, ab125913). Slides were incubated with streptavidin coupled with horseradish peroxidase (HRP), and colour developed using diaminobenzidine (DAB) and DAB Enhancer (Leica).
6
Quantifying Muscle Fiber Types
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Frozen sections (10 μm thickness) of 4-week-old EDL muscles were blocked with M.O.M. (Vector Laboratories, catalog MKB-2213) for 1 hour at room temperature. Sections were incubated with the following antibodies from DSHB: myosin heavy chain type I/IgG2b (catalog BA-D5), myosin heavy chain type IIA/IgG1 (catalog SC-71), and myosin heavy chain type IIB/IgM (catalog BF-F3) at 1:100 dilution, and laminin (MilliporeSigma, L9393) at 1:30 dilution for 45 minutes at 37°C in PBS-1% BSA. After washing with PBS, secondary antibodies (anti–mouse IgG2b Alexa 405, –IgG1 Alexa 488, –IgM Alexa 594, and anti–rabbit IgG Alexa 647) were added for 30–40 minutes at 37°C at 1:200 dilution (Jackson ImmunoResearch). Following washing with PBS, slides were mounted with ProLong Diamond Anti-fade Mounting Medium (Thermo Fisher Scientific, P36970). NIS-Elements Viewer software was used to trace cell boundaries, determine CSA, and analyze fiber type staining. A total of 33,635 WT and 23,362 heterozygous KI fibers were analyzed.
7
Immunofluorescence Staining of Paraffin-Embedded Liver Sections
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Paraffin-embedded liver sections were used for performing the immunofluorescence staining. The sections were deparaffinized as per the standard protocol. Unmasking was done by boiling the sample in 1× citrate buffer in microwave oven for 10 min. Blocking was done using 10% BSA, 5% goat serum, and 5% mouse IgG (MKB-2213, Vector Labs) in phosphate-buffered saline (137 mM sodium chloride, 3 mM potassium chloride, 7 mM disodium hydrogen phosphate, and 11 mM dipotassium hydrogen phosphate, pH 7.4). Primary antibodies used for double staining; anti-rabbit LC3B, 1:100 (Proteintech, 18725-1-AP), and anti-rabbit PLIN2, 1:200 (Proteintech, 15294-1-AP), anti-mouse LAMP1, 1:100 (Genetex, GT25212), p62, 1:100 (Proteintech, 18420-1-AP). Fluorochrome-conjugated secondary antibodies, Alexa-488 or 546 (Invitrogen,) were used. Nucleus was stained with DAPI. Cover slips were mounted on using FluoromountTM aqueous mounting medium (Sigma, F4680). All the images were acquired with Fluorescent microscope (Leica, Leitz, and DMR3) mounted with Nikon camera (Coolpix 5400) and prepared with Adobe Photoshop 7.0.
8
Laminin Immunostaining and Fiber Measurement
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Gastrocnemius muscles were quickly dissected, mounted in 9% Tragacanth gum (Sigma, G1128), frozen in liquid nitrogen-cooled isopentane, and kept at -80 °C. 10 µM cryosections were blocked with mouse on mouse (M.O.MTM) blocking reagent (MKB-2213, Vector Laboratories) before overnight incubation at 4 °C with anti-laminin (L9393, Sigma) primary antibody in DPBS buffer supplemented with 0.5% BSA (A7030, Sigma). The next day, slides were washed in DPBS and stained with anti-rabbit Far Red-Alexa Fluor 647 (711-175-152, Jackson ImmunoResearch) secondary antibody, in DPBS buffer supplemented with 0.5% BSA, for 1 h at 37 °C. After washing in DPBS, slides were mounted in Fluoromount G medium (FP-483331, Interchim, Montluçon, France). Whole muscle section images were acquired at a 10x magnification with a wide-field fluorescence video-microscope (Video Microscope Cell Observer, ZEISS, Oberkochen, Germany). Mosaics were then stitched (Zen 2.3 lite, Zeiss). For each sample, altered fibers were manually removed. Segmentation of all muscle fibers from a cryosection was performed using Cellpose39 (link),40 (link). Mean fiber cross-sectional area was obtained using a self-developed Python script using all muscle fibers from each section.
9
Immunohistochemical Analysis of Ki-67 and FTO
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The paraffin-embedded sections were deparaffinized and treated with (pH 6.0) citric acid buffer for retrieval of antigens, followed by 20 min incubation in a pressure cooker at 104°C and 10 min cooling at room temperature. The sections were then blocked with a mouse IgG blocking agent (#MKB-2213; Vector Labs) for 60 min and quenched with 0.03% H2O2 and sodium azide for 5 min. Subsequently, the sections were incubated with primary mouse monoclonal anti-Ki-67 antibody (1:100, #VP-K452; Vector Labs) or rabbit polyclonal anti-FTO antibody (1;1,000, #PA5-84785; Thermo Fisher Scientific) for 60 min, followed by 20 min incubation with the anti-mouse horseradish peroxidase (HRP)-labeled secondary antibody (K4007, Dako Envision + System; Dako, Carpinteria, CA, USA). After visualization using DAB + chromogen (ab64238; Abcam) for 5 min, the sections were observed with microscopy, and the positive cells in each field of view were counted.
10
Immunofluorescence Staining of Mouse Brain Tissue
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Mice were sacrificed 7 and 35 days post‐dMCAO. Coronal slices (25‐μm‐thick) were prepared for immunofluorescence staining as previously described.18 The following primary antibodies were used: rabbit anti‐NeuN (EMD Millipore), rabbit anti‐microtubule‐associated protein 2 (MAP2; sc‐20,172; Santa Cruz), mouse anti‐200kD neurofilament heavy (NF200; MAB5262; Millipore Sigma), rabbit anti‐beta‐amyloid precursor protein (β‐APP; 512700; Fisher Scientific), rabbit anti‐myelin basic protein (MBP; ab5622; Abcam), goat anti‐Iba1 (ab5076; Abcam), goat anti‐CD206 (AF2535; R&D Systems), rat anti‐CD16 (553142; BD Pharmingen), and mouse anti‐SMI‐32 (ab50761; Abcam). Samples were blocked in 5% normal donkey serum (Jackson ImmunoResearch) to reduce nonspecific binding. When a mouse primary antibody was used, a mouse‐on‐mouse (M.O.M) blocking reagent (MKB‐2213; Vector Laboratories) was used to avoid cross‐reactivity. Donkey Cy3‐ or DyLight 488‐conjugated secondary antibodies (Jackson ImmunoResearch) were used. Images were obtained using an Olympus FluoView FV1000 confocal microscope (Olympus America) and analyzed with ImageJ. Cell numbers were calculated from two random microscopic fields in each mouse brain.
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