Tumor tissues were fixed with 10% NBF overnight, embedded in paraffin, and sectioned according to standard tissue-processing protocol. Formalin-fixed paraffin-embedded tissue sections were stained with hematoxylin and eosin or used for immunohistochemical staining. For immunohistochemistry, the section slides were antigen retrieved (0.01 M Citrate buffer, pH 6.0), stained with indicated primary antibody for 2 h, and then detected by VECTASTAIN Universal Quick HRP Kit (Peroxidase) (Vector laboratory) with DAB kits (Abcam). Hematoxylin staining was used to show nuclear details. We stained Oct4 expression in lymphomas from 40 mice, teratomas in testis tumors from 11 mice, sarcomas from 14 mice, and normal thymi from 6 mice. We counted 1000 lymphomas cells under microscope to obtain the ratio of Oct4+ in lymphomas after Oct4 staining. Six lymphomas were used to analyze the ratio of Oct4+ cells.
Vectastain universal quick hrp kit
Manufactured by Vector Laboratories
Sourced in United States
The VECTASTAIN® Universal Quick HRP Kit is a ready-to-use system for the detection of target antigens in immunohistochemical and immunocytochemical applications. The kit includes a universal biotinylated secondary antibody and a preformed avidin-biotinylated horseradish peroxidase (HRP) complex for a sensitive and rapid detection method.
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Lab products found in correlation
Dab peroxidase hrp substrate kit, by Vector Laboratories (2 mentions)
Dab kit, by Abcam (1 mentions)
Cd8 32 m4 sc 1177, by Santa Cruz Biotechnology (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Anti aldh1a1 antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Evos fl auto imaging system, by Thermo Fisher Scientific (1 mentions)
Vector dab, by Vector Laboratories (1 mentions)
Anti mmp 9, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Dab 3 3 diaminobenzidine, by Merck Group (1 mentions)
Phospho sapk jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
8 protocols using vectastain universal quick hrp kit
1
Immunohistochemical Analysis of Oct4 in Murine Tumors
2
Histopathological Analysis of Tissue Samples
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Tissues and organs were obtained and fixed in 10% neutral formalin, embedded in paraffin, sectioned (5 μm thickness) and stained with H&E (Sigma‐Aldrich). Histopathological analysis was done by an external veterinarian pathologist (National professional certificate 2593012). Immunohistochemistry was done using CD4; MT310 sc‐19641 and CD8; 32‐M4 sc‐1177 (Santa Cruz Biotechnology) primary antibodies, adding the universal biotinylated secondary antibody (VECTASTAIN Universal Quick HRP kit; Vector Laboratories, Burlingame, CA, USA) following the manufacturer instructions, and developed with diaminobenzidine substrate (ImmPACT DAB; Vector Laboratories). Finally, hematoxylin‐counterstained slides were coverslipped using resin as mounting solution and observed under the microscope.
3
Immunostaining of Tumor Tissues
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Anti-ZIP4 antibody (Proteintech) anti-HDAC4 (D15C3) antibody (Cell Signaling Technology), and anti-ALDH1A1 antibody (Sigma) were used as primary antibodies (1:100, 4 °C overnight). A VECTASTAIN® Universal Quick HRP Kit (PK-7800) and a Vector NavaRED Substrate Kit Substrate Kit (SK-4800, Vector Laboratories, Burlingame, CA, USA) were used for detection. Tumor tissues from different groups of mice were examined in immunohistochemistry (IHC) staining. For IF Staining, Alexa Fluor antibodies (PE Donkey anti-Rabbit Cat.Log# 406421 and FITC Donkey anti-Goat Cat.Log# sc-3853) were used as secondary antibodies. Nuclei were counterstained using 4′, 6-diamidino-2-phenylindole (DAPI; Santa Cruz Biotechnology). The staining was analyzed using a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands).
4
Quantifying SUR1 Expression in Cervical Cancer
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A cervical cancer tissue microarray (TMA) containing 39 cases of cervical cancer and 9 cases of normal cervical tissue (in duplicate) was purchased from GeneTex, Inc. (GTX21468). Slides were deparaffinised in xylene, rehydrated in a graded series of ethanol solutions and subjected to antigen retrieval in citric acid. Slides were blocked in normal serum and incubated in primary antibody (SUR1 (75-267, Antibodies Inc.)) overnight at 4 °C. Slides were then processed using the VECTASTAIN® Universal Quick HRP Kit (PK-7800; Vector Laboratories) as per the manufacturer’s instructions. Immunostaining was visualised using 3,3′-diaminobenzidine (Vector® DAB (SK-4100; Vector Laboratories)). Images were taken using an EVOS® FL Auto Imaging System (ThermoFisher Scientific) at ×20 magnification. SUR1 quantification was automated using ImageJ with the IHC Profiler plug-in [88 (link), 89 (link)]. Histology scores (H-score) were calculated based on the percentage of positively stained tumour cells and the staining intensity grade. The staining intensities were classified into the following four categories: 0, no staining; 1, low positive staining; 2, positive staining; 3, strong positive staining. H-score was calculated by the following formula: (3 × percentage of strong positive tissue) + (2 × percentage of positive tissue) + (percentage of low positive tissue), giving a range of 0–300.
5
Paraffin Immunohistochemistry Staining Protocol
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Slides were heated at 55 °C for 10 min, then merged in xylene for 5 min, followed by successive dewaxing processes (2 × xylene, 100% ethanol, 75% ethanol, 50% ethanol, 5 min each), for immunohistochemistry staining on paraffin sections. The slides were then microwaved for 10 min to boil in 10 mM citrate solution for antigen retrieval before being cooled to room temperature. The VECTASTAIN Universal Quick HRP Kit (PK-7800; Vector Laboratories, Table 2 ) was used for the following procedures by the manufacturer's instructions. Staining development was carried out using a DAB Peroxidase (HRP) Substrate Kit (SK-4100; Vector Labs, Table 2 ). The Axio Imager A2 microscope was used to obtain images.
6
Immunohistochemical Analysis of MMP-9 and TNF-α
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Slides were deparaffinized, rehydrated, and immersed in hydrogen peroxide (3%) for 15 min to neutralize endogenous peroxidase. Sections were then washed in phosphate-buffered saline (PBS) and submitted to antigen retrieval using a citrate buffer solution, pH 6, heated to 60 °C for 20 min. Unspecific antigen cross-reaction was ascertained using PBS containing (3%) albumin bovine serum for 20 min. Afterwards, slides were incubated with antibody anti-MMP-9 (1:200) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and TNF-α (1:200) (Santa Cruz Biotechnology Inc., USA) in a humidified chamber at 4 °C overnight. Sections were incubated using the VECTASTAIN® Universal Quick HRP Kit (Vector Labs., Burlingame, CA, USA) detection system, revealed with DAB (3,3-diaminobenzidine) (Sigma-Aldrich, St. Louis, MO, USA), and counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA).
7
Quantifying Phospho-JNK in Cervical Cancer Tissue
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A cervical cancer TMA containing 39 cases of cervical cancer and 9 cases of normal cervical tissue (in duplicate) were purchased from GeneTex, Inc. (GTX21468). Slides were deparaffinised in xylene, rehydrated in a graded series of ethanol solutions and subjected to antigen retrieval in citric acid. Slides were blocked in normal serum and incubated in primary antibody (Phospho-SAPK/JNK (Thr183/Tyr185) (81E11; 4668, Cell Signalling Technology (CST))) overnight at 4 °C. Slides were then processed using the VECTASTAIN® Universal Quick HRP Kit (PK-7800; Vector Laboratories) as per the manufacturer’s instructions. Immunostaining was visualised using 3,3’-diaminobenzidine (Vector® DAB (SK-4100; Vector Laboratories)). Phospho-JNK immunostaining quantification was automated using ImageJ with the IHC Profiler plug-in [88 (link)]. Histology scores (H-score) were calculated based on the percentage of positively stained tumour cells and the staining intensity grade [89 (link)]. The staining intensities were classified into the following four categories: 0, no staining; 1, low positive staining; 2, positive staining; 3, strong positive staining. H score was calculated by the following formula: (3 × percentage of strong positive tissue) + (2 × percentage of positive tissue) + (percentage of low positive tissue), giving a range of 0–300.
8
Immunohistochemistry and Immunofluorescence Protocols
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For immunohistochemistry staining on para n sections, slides rst were heated at 55℃ for 10 min and then merged into xylene for 5 min, followed by consecutive dewaxing steps (2× xylene, 100% ethanol, 75% ethanol, 50% ethanol, 5 min each). Then boiled the slides in 10mM citrate buffer by microwave for 10 min for the purpose of antigen retrieval, cooling the slides in room temperature. The subsequent processes were performed with the VECTASTAIN Universal Quick HRP Kit (PK-7800; Vector Laboratories) following the manufacturer's instructions. DAB Peroxidase (HRP) Substrate Kit (SK-4100; Vector Laboratories) was used for staining developing. Images were acquired by an Axio Imager A2 microscope.
For immuno uorescence staining of culture cells, 100,000 cells were seeded on coverslips in 24-well plates. Next, cells were washed with PBS and xed by 4% PFA for 10 min. Then cells were blocked with 5% BSA, 0.2% Triton X-100 in PBS for 1 hour and incubated with primary antibody in blocking buffer at 4℃ overnight. Next day, cells were incubated with secondary antibody for 2 hours in room temperature in dark, followed by incubating with DAPI for 20 min, and then coverslips were washed and mounted in antifade mounting medium. The uorescence images were acquired by using CONFOCAL microscope with a 25×or 63× objective and analyzed with ImageJ Software.
For immuno uorescence staining of culture cells, 100,000 cells were seeded on coverslips in 24-well plates. Next, cells were washed with PBS and xed by 4% PFA for 10 min. Then cells were blocked with 5% BSA, 0.2% Triton X-100 in PBS for 1 hour and incubated with primary antibody in blocking buffer at 4℃ overnight. Next day, cells were incubated with secondary antibody for 2 hours in room temperature in dark, followed by incubating with DAPI for 20 min, and then coverslips were washed and mounted in antifade mounting medium. The uorescence images were acquired by using CONFOCAL microscope with a 25×or 63× objective and analyzed with ImageJ Software.
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