Immunohistochemical staining was conducted based on the procedures in our previous report [4 (link)]. Specifically, the overnight incubation (4 °C) of testicular tissue sections was executed by virtue of the rabbit polyclonal antibody against ZO-1 (diluted at 1:100; bs-1329R; Bioss) or LC3 (1:100 dilution; 14600-1-AP, Proteintech) in combination with the mouse monoclonal SOX9 antibody (diluted at 1:100; ab76997; abcam, an SC-specific marker). Later, the tissue sections, washed 3 times in PBS, were cultured for 45 min using the FITC-coupled goat anti-rabbit IgG (H+L) antibody (1:200 dilution; HS111, TransGen, Beijing, China) and the PE-labeled goat anti-mouse IgG (H+L) antibody (1:200 dilution; HS221, TransGen, Beijing, China). Lastly, the cell nuclei received DAPI staining based on the previously described methods, followed by observation and photography of the sections under the Olympus-DP73 optical microscope (Tokyo, Japan).
Ab76997
Manufactured by Abcam
Sourced in United Kingdom
Ab76997 is a lab equipment product offered by Abcam. It is a device designed for specific laboratory applications. The core function of this product is to perform a particular task or procedure within a controlled laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Ab3778, by Abcam (2 mentions)
Ab150062, by Abcam (2 mentions)
Ab150107, by Abcam (2 mentions)
Ab177487, by Abcam (2 mentions)
Cm3050, by Leica camera (2 mentions)
F6057, by Merck Group (2 mentions)
Fv3000, by Olympus (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Bs 1329r, by Bioss Antibodies (1 mentions)
Hs111, by Transgene (1 mentions)
Dp73 optical microscope, by Olympus (1 mentions)
Ab32562, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Z031129, by Agilent Technologies (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Immu mount, by Thermo Fisher Scientific (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Ab185430, by Abcam (1 mentions)
11 protocols using ab76997
1
Immunohistochemical Analysis of Testicular Tissue
2
Immunofluorescence Staining of Cultured Cells
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Cells were plated in 12‐well Corning® (Corning, NY, USA) plates using coverslips (12 mm Ø) and fixed for 15 min in 4% paraformaldehyde when highly confluent. Then, cells were permeabilized in PBS 1x‐0.1% Triton and blocked using PBS 1x‐10% Goat serum for 30 min. Primary antibodies SOX9 (1 : 100, ab76997; Abcam, Cambridge, UK), smooth muscle actin (SMA, 1 : 100, RB‐9010‐R7; ThermoFisher Scientific, Waltham, MA, USA), EGFR (1 : 50, ab32562; Abcam), p75 (1 : 100, AB‐N07; ATSbio, Carlsbad, CA), and S100B (1 : 1000, Z031129; Dako, Glostrup, Denmark) were diluted in PBS‐1% Goat serum and incubated overnight at 4 °C. Secondary antibodies Alexa Fluor 488 goat anti‐mouse (1 : 1000, A11029; Invitrogen, Waltham, MA, USA), Alexa Fluor 488 donkey anti‐rabbit (1 : 1000, 711‐545‐152; Jackson ImmunoResearch, Philadelphia, PA, USA), and Alexa Fluor 568 goat anti‐rabbit (1 : 1000, A11036; Invitrogen) were diluted in PBS‐10% Goat serum and incubated for 1 h at RT. After washing three times with PBS 1x, DAPI diluted in PBS (1 : 1000, 62248; ThermoFisher Scientific) was added for 10 min and then washed three times, and finally, coverslips were mounted in Immu‐Mount (9990402; ThermoFisher Scientific). Images were acquired using a Nikon Eclipse 80i fluorescence microscope with nis ‐Elements Microscope Imaging Software and analyzed using imagej fiji software (Lexington, KY, USA).
3
Chondrogenic Differentiation of PCL Scaffolds
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The following reagents were used in this study: PCL (average molecular weight Mn = 45.00 kDa; Sigma-Aldrich, CA, United States), a-DMEM, DMEM/F12 (HyClone, UT, United States), fetal bovine serum (FBS; Gibco, NY, United States), TRITC and FITC phalloidin (R415 and A12379, Invitrogen, CA, United States), 4,6-diamidino-2-phenylindole (DAPI, C0060, MaoKang, Shanghai, China), cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan), primary antibodies anti-collagen type II (COL II; ab185430), anti-collagen type I (COL I; ab6308), anti-aggrecan (AGC; ab3778), SOX-9 (SOX9; ab76997; Abcam, Cambridge, United Kingdom), Alexa Fluor 555-conjugated anti-mouse antibody (A32727, Invitrogen, CA, United States), RNeasy mini kit (Qiagen, Hilden, Germany), SuperScriptTM III Reverse Transcriptase (Bimake, Shanghai, China), and Sirius red solution, Safranin, and fix-green solution (YifanBio, Shanghai, China).
4
Immunofluorescence Staining of Limb Sections
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The tissue sections were deparaffinized and rehydrated using the following steps: 45 min in UC, min in 100% EtOH, 2min in UC, 2min in 100%EtOH, 2min in UltraClear, 5min in 100% EtOH, 5min in 90% EtOH, 5min in 70% EtOH, 15min in H2O bidest. The slides were then placed in DAKO buffer, pH9 and heated in the microwave for 3min twice. The samples were then allowed to stand in the hot buffer for 30min at RT cool down. The tissue was then permeablized in 0.2% of TritonX in PBS for 15min RT and blocked with 5% goat serum/0.2% Tween in PBS for 1h at RT. Limb sections were stained for the following in blocking solution overnight at 4°C—(rabbit-anti-)phospho-Smad1/5/8 (CST-9511L, 1:200), (mouse-anti-)Sox9 (abcam ab76997, 1:150), (rabbit-anti-)Caspase3 (CST-96645, 1:200) and (mouse-anti-)Brdu (Roche-11170376001, 1:50). This was followed by incubation with the secondary antibody in blocking solution for 30 min at RT. For phosphor-Smad1/5/8 an additional tyramide signal amplification step was performed using Tyramide Signal Amplification kit (Perkin Elmer) as per the manufacturer’s instructions. The slides were covered in Fluoromount G and imaged under the fluorescence microscope (Zeiss Axiovert 200).
5
Optical Fiber Verification and Inflammatory Response Analysis
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To verify the proper placement of the optical fibers (fig. S21), animals are deeply anesthetized and transcardially perfused with 0.1 M PBS, followed by 4% paraformaldehyde-borate fixative (pH 7.4) for 20 min. Brains are removed and then postfixed in the same fixative overnight at 4°C, followed by cryoprotection in 10, 20, and 30% sucrose solution at 4°C before being processed for immunocytochemistry. Brains are sliced into 35-μm sections with a cryostat microtome (CM3050 S, Leica). The sections are collected into four serially ordered sets of sections in PBS. For histology examination, the brain sections are washed in PBS and coverslipped with VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (F6057, Sigma-Aldrich). To verify the inflammatory response (fig. S13), after an initial blocking step (3% normal donkey serum in 0.01 M PBS containing 1% Triton X-100), the sections are incubated with the following primary antibodies for 24 hours at 4°C: rabbit anti-NeuN (1:1000; ab177487, Abcam) and mouse anti-SOX9 (1:1000; ab76997, Abcam). The sections are washed and incubated with Alexa Fluor 647 donkey anti-mouse immunoglobulin G (IgG; 1:1000, ab150107, Abcam) and Alexa Fluor 555 donkey anti-rabbit IgG (1:1000, ab150062, Abcam) for 2 hours at approximately 25°C. The fluorescence images are captured using a confocal microscope (FV3000, Olympus).
6
Multimarker Fluorescent Imaging of Liver
7
Immunocytochemical Analysis of Cell-Hydrogel Constructs
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Cell-hydrogel constructs were prepared for immunocytochemistry by first fixing cells in 10% buffered formalin for 1 h, permeabilizing in Triton X-100 for 30 min, and blocking non-specific binding sites with 3% BSA in PBS for 1 h at room temperature. Next, 1-day constructs were incubated with Sox9 primary antibody (abcam, ab76997, 1:200) and 7-day constructs were incubated with aggrecan primary antibody (abcam, ab3778, 1:50) at 4 °C for 16 h. Hydrogels were then rinsed five times with PBS and incubated with secondary antibody (Invitrogen, AlexaFluor 488 goat anti-mouse, 1:200) for 2 h at room temperature. Samples were then rinsed five times with PBS and incubated with DAPI (Life Technologies, 1:500) for 30 min to visualize nuclei. Using a Leica TCS SP8 confocal microscope with a motorized x-y stage, high-resolution 3D tile scan images of the top 100 µm of hydrogels were acquired with a step-size of 4 µm, which were used for subsequent analysis.
8
Comprehensive Tissue Harvesting and Staining Protocol
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Embryo limbs were harvested at desired ages, and fixed in 4% paraformaldehyde for 1 day at room temperature, then switched to 30% sucrose/PBS overnight before they were frozen, embedded, and sectioned. Adult tissues were harvested at desired ages, and fixed in 4% paraformaldehyde for 1 day at room temperature, then decalcified in 10% w/v EDTA (pH 7.4) for 20 days before the joints were embedded in paraffin. Sagittal joint sections were processed for H&E and safranin O‐fast green staining. For immunofluorescence, the following antibodies were used: GFP (Beyotime, AG281; Cell Signaling Technology, 255S), osteocalcin (Millipore, AB10911), Sox9 (Abcam, ab76997), dystrophin (Abcam, ab15277), Mhc (DSHB, MF‐20), Myh3 (DSHB, F1.652), and Runx2 (Sigma, HPA022040); secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 546 or Alexa Fluor 647 or Alexa Fluor 594 fluorescent dyes (Thermo Fisher Scientific) were used for immunofluorescent staining. The stained specimens were photographed digitally under confocal microscope (Nikon A1R).
9
Immunofluorescence for Protein Localization
10
Immunofluorescence Staining of Neural Cells
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Animals were deeply anesthetized, then transcardially perfused with 0.1 m phosphate‐buffered saline (PBS), followed by 4% paraformaldehyde‐borate fixative (PFA, pH 7.4). Tissues were removed and post‐fixed in the PFA 12–14 h at 4 °C, cryoprotection in 30% sucrose solution before for section making. Tissues were sliced into 30 µm sections with a cryostat microtome (CM3050 S, Leica). Sections were washed by PBS and initial blocking (3% normal donkey serum in 0.01 m PBS containing 1% Triton X‐100), and incubated with the primary antibodies overnight at 4 °C, i.e., Rabbit anti‐NeuN (1:1000; ab177487, Abcam) and Mouse anti‐Sox9 (1:1000; ab76997, Abcam). Then the brain sections were washed and incubated with Alexa Fluor 647 Donkey anti‐Mouse IgG (1:1000, ab150107, Abcam) and Alexa Fluor 555 Donkey anti‐Rabbit IgG (1:1000, ab150062, Abcam) for 1.5 h at room temperature. Last, the brain sections were cover slipped with VECTASHIELD mounting media containing DAPI (F6057, Sigma) and images were captured by a confocal microscope (FV3000, Olympus).
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