Tumours were harvested and fixed in 10% formalin, embedded in paraffin, and cut into 4-mm sections. Deparaffinized tumour sections were treated with 3% H2O2 for 10 min to block endogenous peroxidases and incubated with blocking serum (goat serum) at room temperature for 30 min. Immunohistochemistry was carried out with an anti-IL-24 antibody. After incubation with an anti-mouse secondary antibody, the expression of IL-24 was detected with DAB (Sigma, St Louis, MO, USA) by enhancement with an avidin-biotin reaction ABC kit (Vector Laboratories, Burlingame, CA, USA). Tissue sections stained without primary antibody served as negative controls. The slides were then counterstained with haematoxylin. For evaluation of IL-24-positive fractions, at least 200 cells were counted from 6 different regions and the mean number was determined.
Avidin biotin reaction abc kit
Manufactured by Vector Laboratories
Sourced in United States, China, Germany
The Avidin-biotin reaction ABC kit from Vector Laboratories is a reagent system used for the detection of specific target molecules in biological samples. The kit utilizes the high-affinity interaction between avidin and biotin to amplify the signal, enabling sensitive and reliable detection of the target analyte.
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4 protocols using avidin biotin reaction abc kit
1
Immunohistochemical Analysis of IL-24 Expression
2
Quantitative Real-Time PCR Workflow
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The iQ SYBR Green supermix and iScript cDNA synthesis kits were purchased from Bio-Rad (Hercules, CA). The RNAqueous-4PCR and DNA removal kits were obtained from Ambion (Austin, TX). The avidin-biotin reaction (ABC) kit was ordered from Vector Laboratories (Burlingham, CA). Other chemicals were purchased from Sigma (St. Louis, MO).
3
Immunohistochemical Detection of Cleaved Caspase-3
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The tumor sections were incubated with goat anti-cleaved caspase 3 antibodies (diluted 1:150) (Clone: C92-605, Cell Signaling, Boston, MA, USA). After incubation with an anti-rabbit secondary antibody, signals were detected with DAB (Sigma, Beijing, China) and enhanced with an avidin-biotin reaction ABC kit (Vector Laboratories, Burlingame, CA, USA). And the primary antibodies were omitted in the IgG control. The slides were then counterstained with hematoxylin.
4
Immunohistochemical Analysis of SATB1 Expression
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The harvested tumors were fixed in 10% formalin for 24 h at room temperature, embedded in paraffin and cut into 4-mm sections. Subsequent to the samples being deparaffinized with dimethylbenzene and rehydrated using a graduated alcohol series, endogenous peroxidase activity was blocked using 3% H2O2 for 10 min at room temperature, and the sections were then incubated for 30 min at room temperature with goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to block membranes.
Immunohistochemistry was performed using an anti-SATB1 antibody (cat. no. 611182; GenHunter Corporation, Nashville, TN, USA). Following incubation with an anti-mouse secondary antibody (cat. no. PA174460; dilution, 1:1,000; Abcam Company) at 37°C for 30 min, SATB1 expression was determined using 3,3′-diaminobenzidine (DAB; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and enhanced using an Avidin-Biotin Reaction ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA). The negative controls were tissue sections that had been stained without the primary antibody. The proportion of cells positive for SATB1 was determined using optical microscope by counting a minimum of 200 cells from 6 distinct areas, and then calculating the mean.
Immunohistochemistry was performed using an anti-SATB1 antibody (cat. no. 611182; GenHunter Corporation, Nashville, TN, USA). Following incubation with an anti-mouse secondary antibody (cat. no. PA174460; dilution, 1:1,000; Abcam Company) at 37°C for 30 min, SATB1 expression was determined using 3,3′-diaminobenzidine (DAB; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and enhanced using an Avidin-Biotin Reaction ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA). The negative controls were tissue sections that had been stained without the primary antibody. The proportion of cells positive for SATB1 was determined using optical microscope by counting a minimum of 200 cells from 6 distinct areas, and then calculating the mean.
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