Cells were plated in eight-chamber glass slides. After treatment, cells were fixed in 4% paraformaldehyde. After PBS washing, blocking reagent (Dako) was used to block nonspecific background staining for 24 h. After PBS washing, cells were incubated with the anti-STRA6 antibody (Santa Cruz Biotechnology Inc.) at 4°C overnight. After PBS washing, TRITC- or FITC-conjugated secondary antibody (Lonza, Walkersville, MD) was added to cells. Cell membranes were counterstained with green fluorescence-membrane stain (Invitrogen) or nuclei stain, DAPI (Lonza). Under a fluorescence microscope (400×), the red fluorescence images of STRA6 on cell membranes were visualized.
Blocking reagent
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
Blocking reagent is a chemical solution used in various laboratory techniques to prevent non-specific binding of proteins or other molecules to surfaces or substrates. Its core function is to block or minimize unwanted interactions during assays, immunoassays, or other analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Gene expression hybridization buffer, by Agilent Technologies (2 mentions)
Feature extraction v11, by Agilent Technologies (2 mentions)
Agilent mouse v2 4 44k microarrays, by Agilent Technologies (2 mentions)
Agilent 4 44k v2 microarrays, by Agilent Technologies (2 mentions)
Cy3 conjugated anti α sma, by Merck Group (1 mentions)
C6198, by Merck Group (1 mentions)
Dl 1174, by Vector Laboratories (1 mentions)
Bz 2 analyzer, by Keyence (1 mentions)
Bz x700, by Keyence (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Animal research kit, by Agilent Technologies (1 mentions)
M7240, by Agilent Technologies (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Fluoroshield mounting medium with dapi, by Merck Group (1 mentions)
Antibody diluent solution, by Agilent Technologies (1 mentions)
Sa 5004, by Vector Laboratories (1 mentions)
Cleaved caspase 3 asp175 antibody 9661, by Cell Signaling Technology (1 mentions)
Envision system hrp labeled polymer anti rabbit, by Agilent Technologies (1 mentions)
Aec reagent, by Agilent Technologies (1 mentions)
15 protocols using blocking reagent
1
Immunocytochemistry of STRA6 Protein
2
Quantitative Analysis of Microvascular Structure
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At POD 28, five mice from each group were euthanized to collect quadriceps/adductor muscles from ischemic hindlimbs. Harvested muscles were embedded in OCT compound (Tissue-Tek; Sakura Finetek Japan) and snap-frozen in liquid nitrogen. Frozen muscle sections (5-μm thickness) were air-dried and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. After blocking with a blocking reagent (DAKO) including 1% Triton X-100, sections were incubated at 4 °C overnight with DyLite488-conjugated Lycopersicon esculentum Lectin (1:100; DL-1174, Vector Laboratories) for endothelial cell labeling and Cy3-conjugated anti-α-SMA (1:100; C6198, Sigma-Aldrich) for mural cell labeling. Microvessels were evaluated under a fluorescence microscope (BZ-X700; Keyence). Twenty different fields from four different cross sections were randomly chosen, and the number of microvessels per square millimeter was counted using cell counting software (BZ-II analyzer, Keyence); the average microvessel diameter was also obtained using the same microscopic field.
3
Tissue Preparation and Ki-67 Immunohistochemistry
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The tissue samples were fixed in 10% buffered formalin, dehydrated using different grades of isopropanol and embedded in paraffin. Serial sections of 5 µm were made and used for hematoxylin and eosin staining and Ki-67, staining as described earlier52 (link). Briefly, sections were deparaffinised using xylene and rehydrated by passing the slides in different gradient of ethanol and finally to the water. Antigen retrieval was performed using citrate buffer (pH 6) and inactivation of internal peroxidise activity was done using 3%H2O2 in methanol. Blocking was done using blocking reagent provided by DAKO and the sections were incubated with primary antibody Ki-67(#AbD02531, 1:200) overnight at 4 C and visualized using DAB staining. Hematoxylin was used as counter stain and mounted with DPX.
4
Quantifying Tumor Cell Proliferation
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To determine the fraction of proliferating tumor cells, immunohistochemical staining was done with an antibody against human Ki67 (M7240, DAKO). 10 µm-thick tumor cryosections were fixed with a 4% PFA solution. Further treatment of the slides was performed according to the protocol already described for CD31 staining. The anti-human Ki67 antibody was applied to the sections at a concentration of 0.0016 mg/mL. The primary antibody was pretreated with a biotinylation reagent (Animal Research Kit, DAKO) for 20 min. In addition, the primary antibody was mixed with a blocking reagent (DAKO) for 20 min before administration to the cryosections. After incubation overnight at 4 °C, the avidin-biotin complex was applied to the tumor tissue sections for 30 min, which was later developed with AEC. The applied staining reaction was stopped after complete red staining of the proliferating Ki67 containing tumor cells using distilled water. Counterstaining with hematoxylin was used to improve the identification of the non-proliferating tumor cells. Counting of Ki67-labeled cells in 3 to 4 fields of view imaged at 200× magnification was performed using the ImageJ v145.
5
Immunohistochemical Detection of CD44 and CD24
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Sections (4 μm) were de-paraffinized in xylene, rehydrated for CD44 and CD24 detection. The sections were treated with hydrogen peroxide (H2O2; 3% 10 min) at room temperature (RT), washed in distilled water (DW), treated with microwave heating for 15 min in a citrate buffer (pH 9.0) for antigen retrieval, washed in DW and treated with PBS for 5 min. Non-specific binding was blocked by incubation with blocking reagent (Dako) for 5 min at RT, in a wet chamber. Incubation with a monoclonal antibody against all human CD44 isoforms (1:50 dilution, R&D Systems) or all CD24 isoforms (1:1000 dilution, R&D Systems) was carried out for 35 min at 37°C. The immunoreactivity was detected using the Dako Envision kit with peroxidase activity using 3, 3’-diaminobenzidine (DAB) as substrate. Finally, the sections were counterstained with a hematoxylin, dehydrated, cleared in xylene and mounted with Eukitt (DeltaLab). Immunohistochemistry negative controls were prepared by omitting the primary antibody.
6
Lung Arteriolar Smooth Muscle Thickness
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Both lungs were rapidly excised and preserved with 4% paraformaldehyde. Segments from multiple lobes were mounted in paraffin and 5μm transverse sections were cut and mounted onto glass slides for immunohistology. To evaluate arteriolar smooth muscle thickness, slides were incubated in rabbit anti-smooth muscle α-actin (1:200, Abcam) in blocking reagent (Dako), and subsequently incubated in donkey anti-rabbit Alexa Fluor 546 (1:400, Life Technologies) and mounted onto coverslips using FluoroShield™ with DAPI mounting medium (Sigma). Sections were imaged using the TCS SP5 II confocal microscope (Leica Biosystems, Vista, CA). Vessels with diameters 20–110μm were analyzed for wall thickness index, i.e. the total outer vessel area minus the luminal area, divided by total outer vessel area (5 animals per group; 60–250 vessels per animal counted from 10–15 images, taken randomly throughout lung tissue).
7
PAPP-A Immunohistochemistry in Paraffin Sections
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Immunohistochemistry was performed as previously described8 (link). Briefly, following deparaffinisation and rehydration of paraffin embedded tissue slides, endogenous peroxidise activity was blocked with 3% Hydrogen peroxide. 10 mmol/L citrate buffer was used for antigen retrieval. Blocking reagent (Dako) was used to block nonspecific binding. 1.5 µg/mL concentration of PAPP-A antibody (Sigma Aldrich; HPA 001667) was used with overnight incubation at 4 °C. Secondary anti-rabbit antibody HRP (Dako) incubation was performed for 60 min8 (link). 3-amino-9-ethylcarbazole (AEC) was used as the chromogen. Human placenta provided positive control staining for PAPP-A. Primary antibody substituted with the same concentration of rabbit IgG was used as a negative control. ScanScope XT (Aperio) was used to scan slides. Two independent investigators conducted immunohistochemical reactivity evaluations. PAPP-A expression was grouped into four grades: IHC 3+ , strong staining; IHC 2+, moderate staining; IHC 1+, weak staining and IHC 0, no staining. This method was performed in accordance with the relevant guidelines and regulations.
8
Immunohistochemical Analysis of Ki67 and Cleaved Caspase-3
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Tumors were harvested and fixed in formalin. Four-micrometer-thick sections were prepared from paraffin-embedded sections and rehydrated. Antigen retrieval was performed using Sodium Citrate Buffer (10 mmol/L Sodium Citrate, 0.05% Tween 20, pH 6.0) and Citric Acid Buffer (10 mmol/L Citric Acid, 0.05% Tween 20, pH 6.0) following routine protocols. Nonspecific signal was blocked using Blocking reagent (DakoCytomation). Slides were incubated overnight at 4°C with anti-Ki67 (SP6; ab16667 Abcam) or Cleaved Caspase-3 (Asp175) antibody #9661 (Cell Signaling Technology) antibodies diluted following the manufacturer's recommendation in Antibody diluent solution (DakoCytomation). Endogenous peroxidase activity was quenched using H2O2, and ffter several washes in PBS, EnVision+ System HRP-labeled polymer anti-rabbit were added as per requested by the manufacturer's (DakoCytomation), followed by HRP streptavidin (dilution 1:500, SA-5004, Vector). Slides were quickly washed twice in PBS and incubated in AEC+ reagent and counterstained with Mayer's hematoxylin (DakoCytomation). After washing in PBS, slides were mounted with Vectashield (Vector). Immunostaining was recorded with an AXIO optical microscope (Zeiss) equipped with a color AXIOCAM camera 105 (Zeiss, Oberkochen, Germany) and quantified using ImageJ. 15 fields per tumor (n = 2 per condition) were analyzed.
9
Immunohistochemical Staining of CENPF in FFPE Tissue
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Formalin-fixed paraffin-embedded (FFPE) tissue samples were cut into 4 µm thick sections and dried at 60 ℃ for 30 minutes. After blocking endogenous peroxidase activity using blocking reagent (Dako, Santa Clara, CA, USA) for 5 minutes at room temperature, epitopes were retrieved using pH 9.0 Tris-ethylenediamine tetraacetic acid (EDTA) buffer for 40 minutes at 95 ℃ followed by incubation with anti-CENPF (1:200, Proteintech) antibody at room temperature for 30 minutes. Subsequently, the slides were incubated with peroxidase/diaminobenzidine (DAB)-10 min K5007 from a DAKO EnVisionTM Detection Kit (Dako) for 30 minutes.
10
Genomic Array for Comprehensive DNA Analysis
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