Lungs were inflated to 20 mm H2O with 4% paraformaldehyde, fixed overnight and embedded in paraffin. Five-micron sections were deparaffinized in xylene, rehydrated and washed in PBS. Inflammatory cell infiltration was identified by staining with hematoxylin and eosin and viewing by light microscopy. For α-SMA, endogenous peroxidase activity was quenched by incubating sections in 3% hydrogen peroxide (in PBS) for 10 minutes. Sections were fixed and blocked with PBS containing 0.1.% Triton X-100 and 1% BSA at room temperature for 45 minutes. PECAM expression was identified by incubating with an anti-PECAM antibody (1:50) overnight at 4°C followed by FITC anti-rat IgG (1:2000) for 2 hours at room temperature. 4′, 6-diamidino-2-phenylindole (DAPI) dye was used to stain the nuclei. Immunohistochemistry for α-SMA was performed after antigen retrieval in sodium citrate buffer at 95 C for 30 minutes. The sections were blocked with blocking serum (Santa Cruz) and incubated at room temperature for 2 hours, treated overnight with primary antibody (1:200), followed by biotinylated secondary antibody for 1hour, then incubated with AB enzyme reagent and counterstained with hematoxylin. Slides from at least three different animals were reviewed for each stain.
Blocking serum
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Blocking serum is a laboratory reagent used to reduce non-specific binding in immunoassays. It contains a mixture of proteins that can block non-specific antibody interactions, improving the specificity and accuracy of the assay results.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (1 mentions)
Dab reagent, by Agilent Technologies (1 mentions)
Envision reagent, by Agilent Technologies (1 mentions)
Abc staining system, by Santa Cruz Biotechnology (1 mentions)
Ab34712, by Abcam (1 mentions)
Rabbit abc staining system sc 2018, by Santa Cruz Biotechnology (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
5 protocols using blocking serum
1
Histological Analysis of Lung Inflammation
2
Immunohistochemical Analysis of HS3ST1 in Lung Samples
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The detailed immunohistochemical methods have been reported previously67 (link),68 (link). In brief, immunohistochemical analysis for lung samples was performed on 4 µm sections of paraffin-embedded specimens. After deparaffinization and hydration, the slides were treated with endogenous peroxidase in 0.3% hydrogen peroxide solution in 100% methanol for 30 min, after which the sections were blocked with 1.5% blocking serum (Santa Cruz Biotechnology) in PBS before reacting with anti-HS3ST1 antibody at 4 °C in a moist chamber overnight. The slides were washed and treated with Envision reagent, followed by color development in 3,3′-diaminobenzidine tetrahydrochloride (Dako). The slides were lightly counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted. To avoid non-specific binding, immunizing peptide blocking was performed.
Subcutaneous tumors that formed 12 days after the injection of gefitinib into male nude mice were removed and fixed in 10% buffered formalin for 24 h. Formalin-fixed and paraffin-embedded sections (4 μm) were stained with hematoxylin.
Subcutaneous tumors that formed 12 days after the injection of gefitinib into male nude mice were removed and fixed in 10% buffered formalin for 24 h. Formalin-fixed and paraffin-embedded sections (4 μm) were stained with hematoxylin.
3
Immunohistochemical Analysis of FGFR1 Expression
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IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd). Briefly, after deparaffinization and hydration of the sections, the slides were treated with endogenous peroxidase in 0.3% H2O2 for 30 min, followed by blocking for 2h at room temperature with 1.5% blocking serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in phosphate-buffered saline (PBS). Subsequently, the slides were probed overnight with FGFR1 antibody (1:100) at room temperature in a moist chamber. Then, the specimens were washed three times in PBS and treated with Envision reagent (Dako), followed by color development using DAB reagent (Dako). Finally, the slides were counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted. As a negative control, duplicate sections were immunostained without exposure to primary antibodies. To quantitate the FGFR1 protein expression, the mean percentage of positive tumor cells was determined in at least five random fields in each section at 400× magnification. The intensity of the FGFR1 immunoreaction was scored as follows: 1+, weak; 2+, moderate; and 3+, intense. The samples with >10% positive tumor cells and the staining at 1+, 2+, and 3+ levels were considered as FGFR1 IHC-positive.
4
Immunohistochemical Detection of CD133 and CK20
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Endogenous peroxidases were blocked using 3% H2O2 solution for 30 min followed by incubation with blocking serum (Santa Cruz Biotechnology, CA, USA). Incubation with primary antibody of CD133/1 (1 : 40, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or CK20 (1 : 40, Changdao Technology, Shanghai, China) was performed for 1 h at room temperature. And then, immunodetection was performed by ABC staining system (Santa Cruz Biotechnology, CA, USA). Cells were couterstained with haematoxylin to show the nuclear in detail. Negative controls were reacted with PBS in place of primary antibody of CD133 or CK20 [16 , 23 (link)].
5
Collagen Type II Immunohistochemistry
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Immunohistochemical staining of collagen type II was performed using 5 μm sections. Slides were deparaffinized in xylene and rehydrated with graded ethanol. Samples were preincubated with 5 μg/mL proteinase K (Sigma Aldrich) for 10 min at room temperature followed by 1 mg/mL hyaluronidase (Sigma Aldrich) for 40 min at 37°C. Samples were blocked in 1.5% normal goat blocking serum in PBS for 1 h. Rabbit polyclonal collagen type II antibody (Abcam; ab34712) was diluted 1:200 in PBS containing 1.5% blocking serum (Santa Cruz Biotechnology) and incubated overnight at 4°C. Nonimmune controls underwent the same procedure without primary antibody incubation. The target protein was detected by incubation in rabbit ABC staining system (sc-2018; Santa Cruz) according to the manufacturer's protocol and imaged using a NanoZoomer.
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