AP staining was performed by Vector Blue AP Substrate Kit (Vector). The procedure was conducted in accordance with the instruction.
Vector blue ap substrate kit
Manufactured by Vector Laboratories
Sourced in United States, Denmark
The Vector Blue AP Substrate Kit is a reagent used for colorimetric detection in immunohistochemistry and other related applications. It provides a blue chromogenic reaction when used with alkaline phosphatase-conjugated detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Decloaking chamber, by Biocare Medical (2 mentions)
Immpress ap polymer anti rabbit igg mp 5401, by Vector Laboratories (2 mentions)
Vector red ap substrate kit, by Vector Laboratories (2 mentions)
Clone 1a4, by Agilent Technologies (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Pannoramic 250 flash 2, by 3DHISTECH (1 mentions)
Clone jc70a, by Agilent Technologies (1 mentions)
Dm6000 b microsystems, by Leica (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
6 protocols using vector blue ap substrate kit
1
Vector Blue AP Staining Protocol
2
Immunohistochemical Analysis of PDGFR-β, α-SMA, and CD34
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TMA sections were deparaffinised, rehydrated and washed in distilled H2O. After antigen retrieval in decloaking chamber (Biocare Medical) at 110 °C for 5 min in pH 10.0 retrieval buffer, (for PDGFR-β) or pH 9.0 (for α-SMA), sections were incubated with blocking solution for 30 min and then incubated overnight with antibodies against PDGFR-β (No. 3169 Cell Signaling Technology, Danvers, MA, USA at dilution 1 : 100) or α-SMA (Clone 1A4; Dako, Inc., Denmark at dilution 1 : 300). Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Rabbit IgG MP-5401 or ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and developed with Vector Blue AP Substrate Kit (SK-5300, Vector Laboratories).
To destroy alkaline phosphatases activity, sections were subsequently heated in decloaking chamber at 95 °C for 5 min, in pH 9.0 buffer, and then incubated with blocking solution for 30 min. Primary antibody against CD34 (Clone JC70A) at a 1 : 100 dilution was added for overnight incubation at 4 °C. Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories) for 1 h at room temperature and developed with Vector Red AP Substrate Kit (SK-5100, Vector Laboratories).
To destroy alkaline phosphatases activity, sections were subsequently heated in decloaking chamber at 95 °C for 5 min, in pH 9.0 buffer, and then incubated with blocking solution for 30 min. Primary antibody against CD34 (Clone JC70A) at a 1 : 100 dilution was added for overnight incubation at 4 °C. Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories) for 1 h at room temperature and developed with Vector Red AP Substrate Kit (SK-5100, Vector Laboratories).
3
Alkaline Phosphatase Assay for mESCs
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One thousand cells were grown on gelatin-coated glass coverslips for 5 days to form colonies at low density. The cells were seeded in regular mESC medium and allowed to adhere to the coverslips, and then after 12 to 16 hours, the medium was exchanged with either regular mESC medium or with medium containing 250 μM IAA. At day 5, coverslips were washed with 100 mM tris (pH 8) before AP activity assay, which was performed using the VECTOR Blue AP Substrate Kit (Vector Laboratories, VECSK-5300) according to the manufacturer’s instructions. After AP staining, colonies were fixed by incubating with Histofix solution (4% formaldehyde) overnight. The next day, coverslips were washed with 1× PBS and mounted onto glass slides using Fluorescence Mounting Medium (Dako, S3023) and left to dry for at least 16 hours. Images from representative colonies were acquired with a bright-field microscope (Axioplan 2) using a 10× objective. The Pannoramic FLASH 250 II (3DHISTECH) wide-field microscope was used to scan whole slides to obtain sufficient images for quantification of staining intensity and morphology parameters. The images obtained from scanning whole slides were processed with a custom workflow created by T. Lendl (IMBA/IMP BioOptics Core) using Definiens Developer image analysis software.
4
Dual Immunohistochemical Staining for PDGFR-β and CD34
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Four-micrometer-thick sections were de-paraffinised, rehydrated and rinsed in distilled H2O. The antigen retrieval with boiling in pH 10.0 retrieval buffer was performed in decloaking chamber (Biocare Medical) at 110 °C for 5 min. Sections were then incubated with blocking solution for 30 min and with PDGFR-β rabbit monoclonal antibody (#3169, Cell Signaling Technology, Danvers, MA), 2 µg/ml at dilution 1:100 overnight. The sections were incubated with the polymer system (ImmPRESS™-AP Polymer Anti-Rabbit IgG MP-5401) for 1 h at room temperature and developed with Vector® Blue AP Substrate Kit (SK-5300, Vector Laboratories, Burlingame, CA). To inactivate alkaline phosphatase reagents, the sections were heated in decloaking chamber at 95 °C for 5 min, in pH 9.0 solution. This was followed by an incubation with blocking solution for 30 min and with anti-CD34 (Clone JC70A; Dako, Inc., Denmark) at dilution 1:100 overnight. Sections were then incubated with polymer system (ImmPRESS™-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories, Burlingame, CA) for 1 h at room temperature, and developed with Vector® Red AP Substrate Kit (SK-5100, Vector Laboratories, Burlingame, CA).
5
IHC Staining of Tissue Microarray
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IHC staining was performed on formalin-fixed, paraffin-embedded tissue microarray (TMA) as described previously.22 (link) The IHC antibodies are listed in online supplementary table 1 . In brief, the slides were baked at 60°C for 6 hours, deparaffinized in xylene (three times, 15 min each) and rehydrated in graded alcohol. Next, the slides were immersed in sodium citrate buffer (0.01 M sodium citrate buffer, pH=6) for antigen retrieval and then blocked with 3% H2O2 in methanol at 37°C for 30 min. For single IHC staining, the slides were incubated with the primary antibodies at 4°C overnight and visualized by 3,3′-diaminobenzidine (DAB) stain system. For double IHC staining, after being processed as the same of single IHC DAB staining, the slides were incubated with the second primary antibodies at 4°C for 2 hours, and then Vector Blue AP Substrate Kit (Vector Laboratories) was applied. All TMA slides were evaluated under Leica DM6000 B Microsystems by PZ and LC independently, who were blinded to clinical data. The positive cells were enumerated from the representative view of the three sections in high-power field (HPF, ×200 magnification), and the mean value was adopted. The cut-off value was determined by X-tile V.3.6.1 (Yale University). For CD8+ T-cells, the cut-off value was 34 cells/HPF. For TIGIT+ CD8+ cells, the cut-off value was 8 cells/HPF.
6
In Situ Hybridization with LNA-Modified Probes
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In situ hybridisation was performed according to the method described by Budde et al. [28 (link)]. Sections previously immunostained with anti-NeuN (Additional file 1 : Table S2) and detected with a goat anti-mouse-alkaline phosphatase antibody (AP, Dako, Glostrup, Denmark), visualised with Vector Blue AP substrate kit (Vector Laboratories). The hybridisation was performed using a 5′ fluorescein (FAM)-labelled 19mer antisense oligonucleotide that contains locked nucleic acid (LNA) and 2′-O-methyl (2′-O-Me)-RNA moieties (C1q: FAM-TggTccTugAugTuuCcuG, capitals indicate LNA and lower case are 2′-O-Me RNA; Ribo Task ApS, Odense, Denmark). Briefly, sections were pre-hybridised in hybridisation mix (4 M urea in 600 mM NaCl, 10 mM HEPES buffer, pH 7.5, 1 mM EDTA and ×5 Denhardt’s reagent) before probe hybridisation at 55 °C for 45 min in the same solution. Following hybridisation, sections were washed in saline-sodium citrate buffer, and the probe was detected using a sheep anti-fluorescein-AP Fab fragment (Roche Diagnostics GmbH, Penzberg, Germany) and a rabbit anti-sheep immunoglobulin G/horseradish peroxidase (HRP) (Dako). HRP was visualised using Vector NovaRED (red-brown reaction product) prior to permanent mounting (VectaMount, Vector Laboratories).
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