The paraffin-embedded femurs were cut into 5-μm-thick sections on a microtome. After endogenous peroxidase was blocked using 3% H2O2/Mt-OH for 15 min at room temperature, for blocking, normal serum (Gibco, Gaithersburg, MD, USA) was reacted at room temperature for 30 min. The sections were incubated with the primary antibody anti-NFATc1 (1:100) at 4 °C overnight. The tissues were then incubated in secondary antibody (1:100 biotinylated) for 60 min at room temperature. Finally, sections were stained with 3,3′-diaminobenzidine (Vector Labs, Burlingame, CA, USA) and then counterstained with hematoxylin. The stained-tissues were visualized with a light microscope (200×).
Normal serum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Normal serum is a laboratory reagent used as a control sample in various clinical and diagnostic tests. It is derived from pooled human serum and provides a standardized reference material for comparison and validation of test results.
Automatically generated - may contain errors
Lab products found in correlation
Anti c fos, by Santa Cruz Biotechnology (2 mentions)
3 3 diaminobenzidine solution, by Vector Laboratories (2 mentions)
Abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Taq polymerase, by Roche (1 mentions)
Osteo assay surface multiple well plate, by Corning (1 mentions)
Minimum essential medium eagle alpha modification α mem, by Thermo Fisher Scientific (1 mentions)
Raw 264.7 cell, by Korean Cell Line Bank (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti nfatc1, by BD (1 mentions)
Anti cathepsin k, by Santa Cruz Biotechnology (1 mentions)
Anti nfatc1, by Santa Cruz Biotechnology (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab53519, by Abcam (1 mentions)
Emd millipore, by Merck Group (1 mentions)
Eclipse 80i fluorescence microscope, by Nikon (1 mentions)
13 protocols using normal serum
1
Immunohistochemical Analysis of NFATc1
2
Osteoclastogenesis Regulation in RAW 264.7 Cells
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RAW 264.7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Fetal bovine serum (FBS) was obtained from Atlas Biologicals (Fort Collins, CO, USA). Penicillin/streptomycin (P/S), minimum essential medium eagle alpha-modification (α-MEM), and normal serum were purchased from Gibco (Gaithersburg, MD, USA). E2 and ALN were supplied by Sigma-Aldrich (St. Louis, MI, USA). Osteo assay surface multiple-well plates were supplied by Corning Inc. (Corning, NY, USA). Nitrocellulose membranes were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Taq polymerase was purchased from Kapa Biosystems (Woburn, MA, USA). Primary antibodies against anti-β-actin (cat no: 8432) and anti-c-Fos (cat no: sc-447) were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-NFATc1 (cat no: 556602) was supplied by BD Pharmingen, Inc. (San Diego, CA, USA), and secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
3
Immunohistochemical Analysis of Bone Remodeling
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The left femur was fixed in 10% NBF for 2 days, demineralized using 10% EDTA-2Na for 3 weeks, and then dehydrated with ethanol, clarified with xylene, and embedded with paraffin. Paraffin embedded tissue was sectioned on a rotary microtome. Endogenous peroxidase was blocked in 3% H2O2/Mt-OH for 15 min at room temperature. Then, 20 μg/ml proteinase K (Thermo fisher, PA, USA) was used for epitope retrieval for 10 min at 37 °C, for blocking, normal serum (Gibco, Gaithersburg, MD, USA) was reacted at room temperature for 30 min. The tissues were incubated with primary antibody diluted in 0.5% BSA, including anti-NFATc1 (1:100, Santa Cruz Biotechnology, CA, USA), anti-c-fos (1:100, Santa Cruz Biotechnology, CA, USA) and anti-cathepsin K (1:100, Santa Cruz Biotechnology, CA, USA), at 4 °C overnight. The tissues were then stored in 1:100 biotinylated secondary antibody (Vector Labs, Burlingame, CA, USA) for 60 min at room temperature. The tissues were incubated in horseradish peroxidase-streptavidin using ABC kit (Vector Labs, Burlingame, CA, USA) for 30 min at room temperature and stained with 3,3′-Diaminobenzidine solution (Vector Labs). The tissues were counterstained with hematoxylin, dehydrated and mounted. The immunohistochemical stained tissues were observed with a light microscope (100, 200×).
4
Cryosectioning and Immunofluorescence of Liver
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Cryostat sections of liver samples were prepared, fixed in pre-cooled 100% methanol at −20°C, and blocked with 10% normal serum (Gibco) in 1% fish skin gelatin prior to addition of primary Abs and secondary Abs. Tissue sections were counterstained with 400 ng/ml DAPI (Sigma), mounted in media (DAKOCytomation, USA) and analyzed by confocal microscopy. Excitation wavelengths were 405, 488, and 555 nm, and emission wavelengths maxima were 493/519 and 557/574 nm.
5
Immunohistochemical Analysis of NFATc1 and CTK
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Paraffin-embedded tissues were deparaffinized in xylene. Endogenous peroxidases were blocked in 0.3% hydrogen peroxide at room temperature for 15 min and proteinase K (0.4 mg/ml) was used for antigen-retrieval at 37°C for 30 min. The sectioned tissues were incubated with 10% normal serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 1 h to block nonspecific binding, then slides were washed with PBS and incubated at 4°C for overnight with primary antibodies against NFATc1 (1:100) and CTK (1:100). The following day, the slides were washed with PBS and incubated with a secondary antibody (1:100; rabbit; cat. no: BA-1000; Vector Laboratories, Inc.) at 4°C for 1 h. The signal was visualized using an ABC kit (Vector Laboratories, Inc.) and 3, 3′-diaminobenzidine solution (Vector Laboratories, Inc.). The stained tissues were imaged using a light microscope (magnifications, ×100 and ×200).
6
Immunohistochemical Analysis of Snail and N-Cadherin
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Tissues were fixed in 10% neutral-buffered formalin, processed and embedded in paraffin. Tissue sections were deparaffinized and rehydrated in an ethanol series. Sections were blocked for nonspecific binding with 1% normal serum (Thermo Fisher Scientific, Inc.) and incubated with the primary anti-Snail (ab53519; diluted at 1:500; Abcam) and anti-N cadherin (PA5-19486; diluted at 1:300; Thermo Fisher Scientific, Inc.) antibodies overnight at 4°C. Subsequently, immunostaining was developed using 3,3′-diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA, USA) followed by hematoxylin counterstaining (Sigma-Aldrich; EMD Millipore). Immunostaining was visualized using a fluorescence microscope (Eclipse 80i Fluorescence Microscope; Nikon Corporation).
7
Immunostaining of hPDLSCs for Vimentin
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hPDLSCs were cultured in a 24-well plate. At 60% confluence, 0.5 ml 4% paraformaldehyde (cat. no. MFCD00133991; Thermo Fisher Scientific, Inc.) was added into each well for 20 min at room temperature. After washing the cells with PBS (cat. no. 10010031; Thermo Fisher Scientific, Inc.) three times, 0.2% Triton X-100 (cat. no. HFH10; Thermo Fisher Scientific, Inc.) was added to the cells for 30 min, and subsequently, the cells were incubated with 3% H2O2 for 10 min at room temperature. Cells were blocked in 10% normal serum (Thermo Fisher Scientific, Inc.) with 1% BSA (Thermo Fisher Scientific, Inc.) in TBS for 2 h at room temperature. Each well was incubated with 50 µl anti-vimentin (cat. no. ab193555; 1:200; Abcam) antibody for 2 h at 37°C. After washing with PBS three times for 2 min each time, 50 µl goat anti-rabbit IgG H&L preadsorption secondary antibody (cat. no. ab96899; Abcam, 1:20,000) was added to each well for 20 min at 37°C. The cells were then washed with PBS three times. Subsequently, a DAB Reagent kit (cat. no. PW017; Shanghai Sangong Pharmaceutical Co., Ltd.) was used and the cells were observed under a fluorescence microscope at ×400 magnification).
8
Immunohistochemical Analysis of Articular Cartilage
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Coronal disc tissue sections with 6 μm thickness were gently rinsed with PBS and incubated with proteinase K (20 μg/mL, D3001‐2‐5, Zymo Research) for 10 minutes at room temperature (RT). Subsequently, sections were blocked in 5% normal serum (10000 C, Thermo Fisher Scientific, Swedesboro, New Jersey) in PBS‐T (0.4% Triton X‐100 in PBS) or incubated with antibodies against type II collagen (1:100, ab34712, Abcam) in blocking buffer at 4°C for overnight, and then incubated in second antibodies Alexa Fluor 488‐conjugated anti‐rabbit (1:200, A11008, Invitrogen, Carlsbad, California) or Alexa Fluor 647‐conjugated anti‐mouse (1:200, A‐21236, Invitrogen, Carlsbad, California) antibodies for 1 hour at RT. Coverslips were mounted with Fluoroshield (F6057, Sigma‐Aldrich, St. Louis, Missouri).
To quantify the percentage of tdTomato+ cells, multiple fields of Z‐stacked pictures were randomly captured. At least 30 images were measured. The percentage of tdTomato+ cell was calculated from the ratio of tdTomato+ cells over total cells observed in each compartment of each sample (five sections per sample). Six mice were evaluated in each group. Assessments were independently completed by two investigators who were blinded to the treatment or groups. The average percentage of tdTomato+ cells in each sample and group were pooled and calculated by two investigators.
To quantify the percentage of tdTomato+ cells, multiple fields of Z‐stacked pictures were randomly captured. At least 30 images were measured. The percentage of tdTomato+ cell was calculated from the ratio of tdTomato+ cells over total cells observed in each compartment of each sample (five sections per sample). Six mice were evaluated in each group. Assessments were independently completed by two investigators who were blinded to the treatment or groups. The average percentage of tdTomato+ cells in each sample and group were pooled and calculated by two investigators.
9
Histological Analysis of Enpp1 in Mice
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The knee joints of WT and Enpp1−/− mice were harvested and stored in 10% formalin. Then knee joints were decalcified in 0.5 M EDTA (pH 7.4) for 4 weeks at 4 °C, changing EDTA every 2 days. The joints were made into 6-μm sections after embedding with paraffin. After deparaffinizing, the sections were incubated with proteinase K (20 μg/mL, Zymo Research, USA) for 10 min at room temperature and blocked in 5% normal serum (10,000 C, Thermo Fisher Scientific, USA) in PBS-T (0.4% Triton X-100 in PBS). Subsequently, the sections were incubated with antibodies against Enpp1 (1:1000, Bioss Antibodies Inc, Woburn, MA, USA) for 12 h and secondary antibody (Alexa Fluor 488 Labeled Goat Anti-Rabbit IgG, Beyotime, China) for an hour, at 37 ℃. Finally, the cover glass was placed on the section after adding the mounting medium. The images were captured by an inverted fluorescence microscope (Leica) and then the proportion of Enpp1-positive chondrocytes (green fluorescence) was analyzed by ImageJ.
The 6-μm sections described previously were stained with Hematoxylin & Eosin and Safranin O/Fast Green according to standard procedures.
The 6-μm sections described previously were stained with Hematoxylin & Eosin and Safranin O/Fast Green according to standard procedures.
10
Histopathology and Immunofluorescence of Caudal Discs
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For histopathological analysis, tissues were fixed in 10% formalin for 96 h, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for at least one month, embedded in paraffin, and 4–5 µm sections were stained with Safranin O/Fast Green Stain. For immunofluorescence, the caudal disc sections were blocked in 5% normal serum (Thermo Fisher Scientific, 10000 C) in PBS‐T (0.4% Triton X‐100 in PBS) and incubated with the primary antibody. The primary antibodies used were CD11b (1:500, 120772, Absin), OLR1(1:500, Absin, 123947), CD24 (1:200, Proteintech, 10600‐1‐AP). After washed with PBS/1% BSA, the sections were incubated with Alexa‐conjugated antibodies and washed with PBS/1% BSA followed by PBS. The DAPI was used for counterstaining.
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