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Sk 5300

Manufactured by Vector Laboratories
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Sourced in United States

The SK-5300 is a laboratory instrument designed for fluorescent immunohistochemistry and immunocytochemistry applications. It provides a controlled environment for imaging and analysis of fluorescent samples.

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Lab products found in correlation

Axio scan z1, by Zeiss (1 mentions) Cm1950, by Leica (1 mentions) Vectastain abc ap kit, by Vector Laboratories (1 mentions) Osteomeasure software, by OsteoMetrics (1 mentions) Sk 4805, by Vector Laboratories (1 mentions) Mp 5401, by Vector Laboratories (1 mentions) Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Sk 5100, by Vector Laboratories (1 mentions)

6 protocols using sk 5300

1

Histological Analysis of Intervertebral Discs

Cited 2 times
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Frozen calcified sections of WT and N153S discs (L1-L2, Ca1-Ca2) were cut at 10 μm and secured to slides using cryofilm 2C (10 (link)) to maintain the morphology of the mineralized sections (40 (link)). The taped sections were glued to microscope slides using chitosan adhesive and rehydrated prior to imaging. Sections were also stained to detect activity of tartrate-resistant acid phosphatase (TRAP) (1:80, Invitrogen, E6601A) and tissue non-specific alkaline phosphatase (TNAP) (Vector Laboratories, SK5300).
Ottone O.K., Kim C.J., Collins J.A, & Risbud M.V. (2022). The cGAS-STING Pathway Affects Vertebral Bone but Does Not Promote Intervertebral Disc Cell Senescence or Degeneration. Frontiers in Immunology, 13, 882407.
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2

Pluripotency Marker Expression in ESC/iPSC

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ESC or iPSC cultures were fixed in 4% paraformaldehyde (PFA), permeabilized by 1% Triton-X (excluded for cell surface markers) and blocked with 2% BSA and 130 mM glycine in phosphate buffer saline (PBS). After overnight incubation with primary antibodies Oct4 (Santa Cruz Biotechnology Cat# sc-5279 Conc: 1:500), SSEA-4 (Chemicon Cat# MB4303 Conc: 1:250) and TRA-1-60 (Chemicon MAB4360 1:200) followed by thorough washes, a secondary antibody conjugated with Alexa Red (Molecular Probe Cat# A21203 Conc: 1:1000) was used for detection of the primary antibodies. DNA was counterstained with Hoechst 33342 (5 µg/ml Molecular probe Conc 1:10,000). The specimen was examined using an epifluorescent microscope. Alkaline phosphatase assay was performed following the manufacturer’s instructions (Vector Lab Cat# SK-5300).
Moran S., Chi T., Prucha M.S., Ahn K.S., Connor-Stroud F., Jean S., Gould K, & Chan A.W. (2015). Germline transmission in transgenic Huntington’s disease monkeys. Theriogenology, 84(2), 277-285.
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3

Osteochondral Defect Characterization Workflow

Cited 1 time
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Osteochondral units were fixed immediately after μCT scanning for 24 h in 10% neutral buffered formalin. After fixing, samples were infiltrated for 48 h with a 10 % sucrose and 2% PVP solution. Then, samples were embedded in OCT compound and sectioned (18 μm/section) undecalcified using cryofilm until reaching the midplane of the defect using a motorized cryostat (CM1950, Leica) [37 ]. Tape-stabilized, frozen sections were subjected to two rounds of imaging using a Zeiss AxioScan.Z1 digital slide scanner, including (i) fluorochrome label with TO-PRO-3 Iodide nuclear counterstain, and (ii) AP fluorescent staining with Hoechst 3342 nuclear counterstain [38 (link)]. Before AP staining, sections were decalcified for 1 h in 0.92% sodium acetate anhydrous, 1.14% L-(+)-tartaric acid, and 1% glacial acetic acid pH 4.1–4.3. AP staining occurred according to the manufacturer’s protocol (Vector Laboratories, Burlingame, CA, USA, SK-5300).
Zlotnick H.M., Locke R.C., Hemdev S., Stoeckl B.D., Gupta S., Peredo A.P., Steinberg D.R., Carey J.L., Lee D., Dodge G.R, & Mauck R.L. (2022). Gravity-based patterning of osteogenic factors to preserve bone structure after osteochondral injury in a large animal model. Biofabrication, 14(4), 10.1088/1758-5090/ac79cd.
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4

Cartilage Thickness and Osteoarthritis Scoring

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Four and 12 week old control and cKO mice were sacrificed and the femurs were fixed in 10% formalin for 4 days, decalcified, and processed for paraffin sectioning as previously described40 (link). Three comparable sections of the knee joint from each animal were stained with Safranin O and counter stained with Fast Green to visualize the articular cartilage. Measurements of the thickness of the SZ and the MDZ were carried out using the OsteoMeasure software (Osteometrics, Inc. Decatur, GA). OARSI scoring was according to previously described41 (link). Immunohistochemistry was performed using the VECTASTAIN ABC-AP kit (AK-5000, Vector Laboratories, Burlingame, CA) as previously described40 (link). Antibodies and dilutions of each antibody were listed in Supplemental Table 1. Vector Blue was used as the AP substrate (SK-5300, Vector Laboratories, Burlingame, CA).
Cheng S., Pourteymoor S., Alarcon C, & Mohan S. (2017). Conditional Deletion of the Phd2 Gene in Articular Chondrocytes Accelerates Differentiation and Reduces Articular Cartilage Thickness. Scientific Reports, 7, 45408.
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5

Immunohistochemical Analysis of DCLK1+ Cells

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Unstained 5-micron sections derived from the formalin-fixed and paraffin-embedded blocks were deparaffinized and hydrated by routine procedures. Sodium citrate buffer (pH 6.0) was used as the antigen retrieval. The primary antibodies at diluted concentrations were incubated overnight at room temperature. The primary antibodies are listed in Supplementary Table 1. For immunohistochemistry (IHC), the secondary antibodies used were Dako LSAB+system-HRP (universal) and Envision+system-HRP (anti-rabbit and anti-mouse polymer-HRP). For double IHC, anti-rabbit and anti-mouse polymer-AP kits were purchased from Vector (MP-5401 and MP5402), including the substrate kits for red peroxidase (SK-4805), red alkaline phosphatase (SK-5100) and blue alkaline phosphatase (SK-5300). For immunofluorescence (IF) assay, the fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch) were incubated at room temperature for 2 hours. All other procedures were done according to the manufactures’ instructions. The percentage of DCLK1+ cells for each experimental group (ADM, PanIN, IPMN, and normal) was determined by counting DCK1+ cells and total ductal epithelial cells present in the pancreata of ten randomly chosen mice within each group (MT-TGF-α, KP, AKP GEMM, and Cre-negative control), and at least three different sections of each individual pancreas were examined.
Qiu W., Remotti H.E., Tang S.M., Wang E., Dobberteen L., Youssof A.L., Lee J.H., Cheung E.C, & Su G.H. (2018). Pancreatic DCLK1+ Cells Originate Distinctly from PDX1+ Progenitors and Contribute to the Initiation of Intraductal Papillary Mucinous Neoplasm in Mice. Cancer letters, 423, 71-79.
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6

Characterization of Mineralized Disc Sections

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Frozen calcified sections of WT and N153S discs (L1-L2, Ca1-Ca2) were cut at 10 m and secured to slides using cryofilm 2C (10) to maintain the morphology of the mineralized sections (40) . The taped sections were glued to microscope slides using chitosan adhesive and rehydrated prior to imaging. Sections were also stained to detect activity of tartrate-resistant acid phosphatase (TRAP) (1:80, Invitrogen, E6601A) and tissue non-specific alkaline phosphatase (TNAP) (Vector Laboratories, SK5300).
Ottone O.K., Kim C., Collins J.A., & Risbud M.V. (2022). The cGAS-STING pathway affects vertebral bone but does not promote intervertebral disc cell senescence or degeneration.
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