Tissue sections were deparaffinized and rehydrated, endogenous peroxidase activity neutralized and antigens retrieved by boiling in sodium citrate solution (Vector Labs H-3300). Sections were blocked in 2% bovine serum albumin (BSA)/10% normal serum (same species of secondary antibody) and avidin-blocking solution (Vector) in phosphate-buffered saline (PBS), incubated with primary antibody in 2% BSA/10% normal serum and biotin-blocking solution (Vector) in PBS, incubated with biotinylated secondary antibody 1:500 (Vector), incubated with ABC (Vector PK-6100) or ABC-AP reagent (Vector AK-5000), incubated with DAB/substrate/chromogen system (Dako K3467; brown) or AP substrate kit I (Vector SK-5100 reagent, red) and counterstained with hematoxylin (Vector H-3404). Specificity of staining was confirmed by omission of the primary antibody. Images were obtained using a digital microscope (COOLSCOPE, Nikon). Primary antibodies used included anti-β-catenin (Santa Cruz sc-7963, 1:250 dilution), anti-α-SMA (Sigma A2547, 1:200 dilution), anti-Sm22α (Abcam 14106, 1:500 dilution), anti-CD31 (Abcam 28364, 1:50 dilution) and anti-pHH3 (Cell Signaling 9701, 1:50 dilution).
K3467
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The K3467 is a high-performance laboratory equipment designed for scientific research and analysis. It is a precision instrument that provides reliable and accurate measurements for various applications. The core function of the K3467 is to perform advanced data acquisition and analysis tasks, enabling researchers and scientists to gather and process critical information effectively.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (2 mentions)
Biotin blocking solution, by Vector Laboratories (1 mentions)
Avidin blocking solution, by Vector Laboratories (1 mentions)
H 3300, by Vector Laboratories (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Coolscope, by Nikon (1 mentions)
E0354, by Agilent Technologies (1 mentions)
M0701, by Agilent Technologies (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Ab15627, by Abcam (1 mentions)
A11059, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 labeled goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Anti f4 80 mca497ga, by Bio-Rad (1 mentions)
E0466, by Agilent Technologies (1 mentions)
P0397, by Agilent Technologies (1 mentions)
A11058, by Thermo Fisher Scientific (1 mentions)
E0464, by Agilent Technologies (1 mentions)
Ab5694, by Abcam (1 mentions)
M glas, by Merck Group (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
10 protocols using k3467
1
Immunohistochemical Staining of Tissue Sections
2
Quantifying CD45+ Cells in Pancreatic Islets
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Human paraffin-embedded pancreatic tissue slides were received from the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative type 1 diabetes research project sponsored by JDRF. Organ Procurement Organizations partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners/ . nPOD tissue samples are classified as “Non-Human Subjects” according to the University of Florida IRB. Approved by the Basel Ethical Committee, no Ethical Board review was required. Tissue slides were deparaffinized, rehydrated and stained with mouse anti-human CD45 (1:100, overnight at 4 °C; DAKO, M0701) and biotinylated anti-mouse immunoglobulins (1:200, 60 min at room temperature, DAKO, E0354) and visualized with DAB (2 min, DAKO, K3467). Pictures of 30–35 islets per section were obtained and CD45+ cells within and around islets were counted. Intra-vessel CD45+ cells were excluded. Staining, photo acquirement and cell counting was done in a blinded manner and unblinding occurred after counting all samples.
3
GFAP Immunohistochemistry in Rat Tissue
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In all individual rats, an additional series of cryo-sections was used for glial fibrillary acidic protein (GFAP) immunohistochemistry. Sections were fixated in 10% neutral buffered formalin for 10 min followed by rinsing in water. Endogenous peroxidase activity was blocked for 10 min in 1% H2O2 + tris buffered saline (TBS) + 0.3% Triton-X (TX). Blocking of unspecific binding was obtained with 5% swine serum in TBS + 0.3% TX + 1% BSA for 20 min followed by addition of rabbit anti-GFAP antibody (1:300; Z0334; Dako, Denmark) diluted in TBS-T + 1% BSA for 60 min. The reaction was amplified for 30 min using MACH 2 rabbit HRP-polymer (RHRP520, BioCare Medical, USA). Sections were rinsed in TBS before subjected to 3,3′-diaminobenzidine as chromagen (K3467; Dako, Denmark). The evaluation of astrocyte profiles was performed on digitalized slides using the newCAST software (Visiopharm, Hørsholm, Denmark). A region of interest was delineated and a counting frame was placed using systematic uniform random sampling principles. Astrocyte profiles were counted when located within the counting frame. A total number of approximately 50 counting frames was evaluated.
4
Immunohistochemical Analysis of Liver Markers
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Liver sections were incubated with primary antibodies, followed by incubation with biotinylated secondary antibodies. The following primary antibodies were used: anti-STK25 (YSK1; sc-6865; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PCNA (MA5-1158; Invitrogen), anti-Ki67 (14-5698-82; Invitrogen), anti-Gr1 (Ly6C) (ab15627; Abcam, Cambridge, UK), anti-F4/80 (MCA497GA; Bio-Rad, Hercules, CA), anti–collagen I (SAB4500362; Sigma-Aldrich), anti–α-smooth muscle actin (ab5694; Abcam), anti–4-HNE (sc-130083; Santa Cruz Biotechnology), anti-PEX5 (PA5-58716; Invitrogen), and anti-ubiquitin (ab411; Abcam). For immunohistochemical detection, anti-goat IgG (E0466; Dako, Glostrup, Denmark) and anti-mouse IgG (E0464; Dako) secondary antibodies were used, followed by horseradish-peroxidase–conjugated streptavidin (P0397; Dako) and diaminobenzidine staining (K3467; Dako). For immunofluorescence detection, Alexa Fluor-594–labeled goat anti-rat IgG (A11007; Invitrogen), Alexa Fluor-488–labeled rabbit anti-mouse IgG (A11059; Invitrogen), Alexa Fluor-594–labeled donkey anti-goat IgG (A11058; Invitrogen), and Alexa Fluor-594–labeled donkey anti-rabbit IgG (A21207; Invitrogen) secondary antibodies were used. The stained area was quantified in 5 randomly selected microscopic fields (×200) per mouse using ImageJ software.
5
Quantitative Analysis of Tumor Angiogenesis
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Tumor slices were placed at room temperature, fixed in acetone for 10 min and washed twice in TBS with 0.1% Tween 20 (TBS/T). Endogenous peroxidases were blocked with 1% H2O2/methanol, and slices were rinsed in distilled water and TBS/T for 5 min. Tumor sections were blocked with 2% rabbit serum diluted in TBS for 20 min and were rinsed for 5 min in TBS/T. CD31 was labeled with the rat anti-CD31 antibody for 1 hour (#550274 at 1/500; BD Biosciences, Pont de Claix, France) and the rabbit anti-rat antibody for 1 hour (#7077 at 1/750; Cell signaling Technology, Danvers, MA, USA). The labeling was detected with DAB for 5 min (#K3467; Dako, San Antonio, TX, USA) and rinsed with water for 5 min. Cell nuclei were stained with Gill’s hematoxylin for 2–3 s and washed with water 5 for min. Samples were dehydrated with 2 baths of 100% ethanol and one with xylene before the addition of mounting medium (M-GLAS, #1.03973.001; Merck Millipore, Fontenay sous bois, France). Images were taken using the Olympus BX-41 microscope with a 10× objective.
The evaluation of blood vessel number and diameter by CD31-quantification was performed on four fields of view for each tumor using Metamorph software.
The evaluation of blood vessel number and diameter by CD31-quantification was performed on four fields of view for each tumor using Metamorph software.
6
Proliferating Cell Dynamics in Jejunum
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Proliferating cells in the jejunum were labeled with BrdU (ab142567, Abcam, London, UK), which was injected intraperitoneally into mice at 10 mg/kg 2 h before sampling. Briefly, deparaffinized and rehydrated sections were submerged in pH 6.0 sodium citrate buffer and heated to 95 °C for 15–30 min for antigen retrieval. Next, the sections were incubated with peroxidase-blocking solution (S2023, Dako, Carpinteria, CA, USA), followed by incubation with 1% normal goat serum for 30 min at room temperature to block nonspecific binding. Sections were washed with PBS three times and incubated with a primary antibody against BrdU (#5292, Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature, followed by 1 h incubation with a species-specific Envision+ kit (Dako, Carpinteria, CA, USA), according to the manufacturer’s instructions. Liquid DAB + (K3467, Dako, Carpinteria, CA, USA) was used for visualizing. For immunofluorescence staining, anti-Ki-67 antibody (#12202, Cell Signaling Technology, Danvers, MA, USA) and DAPI (D9542, Sigma-Aldrich, St. Louis, MO, USA) were used. For TUNEL staining, In situ Cell Death Detection Kit (#1168-481-7910, Roche, Indianapolis, IN, USA) was used according to the manufacturer’s instructions.
7
Immunohistochemical and Immunofluorescence Staining of Fallopian Tube Tissues
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For immunohistochemical staining, the Fallopian tubal tissues were formalin‐fixed, paraffin‐embedded, sectioned, and mounted onto slides. Tissue sections were deparaffinized and rehydrated. The slides were then incubated in a 3% H2O2 solution to block endogenous peroxidase and rinsed with PBS. The sections were treated with heated antigen retrieval solution containing EDTA and then incubated with 5% bovine serum albumin for 30 min and then with the primary antibody rabbit anti‐TRPV4 at 4 °C for 12 h. After incubation with goat anti‐rabbit secondary antibody for 60 min, the sections were treated with diaminobenzidine (K3467; DakoCytomation, Glostrup, Denmark), counterstained with hematoxylin, dehydrated, and mounted in Distrene dibutyl phthalate xylene (DPX). For immunofluorescence, the tissues were paraformaldehyde‐fixed and then embedded in optimal cutting temperature compound; a 10 μm section was then cut and mounted. The sections were blocked for 1 h at room temperature and incubated with the primary antibody (listed in the supplementary material, Table S1 ) at 4 °C for 12 h. A secondary fluorescent antibody was used after the sections were washed three times with PBS. The sections were mounted with Antifade Mounting Medium containing 4,6‐diamidino‐2‐phenylindole (DAPI; VECTASHIELD, Vector Laboratories, Burlingame, CA, USA).
8
Immunohistochemical Analysis of LSD1 and VDR Expression
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Tissues were fixed in 10% buffered formalin for 24 h prior to processing. Tissues were processed and embedded in paraffin and then sectioned at 5 μm. Slides were deparaffinized in several baths of xylene and then rehydrated in graded alcohols followed by ddH2O. Slides were incubated in 1× pH 6 citrate buffer (Invitrogen, 00-5000) in DAKO PT Link for 20 min. IHC was performed using DAKO Autostainer Plus following manufacturer’s instructions. Slides were incubated in 3% H2O2 for 15 min. To block non-specific binding, tissues were incubated with 10% normal goat serum for 30 min, followed by avidin/biotin block (Vector Labs, SP-2001). Primary antibody LSD1 (Cell Signaling, 2139) or VDR (Thermo Scientific MA1710 (Clone 9A7)) were diluted in 1% BSA solution and incubated for 30 min at room temperature, followed by the biotinylated Goat Anti-Rabbit secondary antibody (Abcam, ab6720) for 15 min. For signal enhancement, ABC reagent (Vector Labs, PK-6100) was applied for 30 min. Slides were then incubated with DAB substrate (Dako, K3467) for 5 min and then counterstained with DAKO hematoxylin for 20 s. Slides were dehydrated through several baths of graded alcohols and xylenes and then coverslipped. Finally, slides were scanned using the Aperio System (Leica).
9
Paraffin-Embedded Tissue Immunostaining
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Paraffin-embedded tissue was stained as published previously (6) . In brief, tissue was rehydrated, and antigen retrieval was performed in a Tris-EGTA buffer (TEG, 10 mM Tris, 0.5 mM EGTA, pH = 9.0). Endogenous peroxidase enzymes were blocked using 0.6% H (K3467, DakoCytomation). Sections were counterstained with hematoxylin. Sections were visualized by light microscopy with an Olympus BX51 microscope.
10
Immunohistochemical Detection of CLDN14
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Paraffin-embedded tissues were stained as previously (21) . In brief, the tissues were rehydrated, and antigen retrieval was performed in a Tris-EGTA buffer (TEG, 10 mM Tris, 0.5 mM EGTA, pH = 9.0). Endogenous peroxidase enzymes were blocked using 0.6% H 2 O 2 and 50 mM NH 4 Cl in PBS was added to block free aldehyde groups. Sections were then incubated with a primary anti-CLDN14 antibody in 0.1% Triton X-100 in PBS overnight at 4°C. The monoclonal antibody against CLDN14 used in this study is directed against the epitope CYRPPAAYKDNRAPSV-TSAAHSGYRLNDYV in the extreme C-terminal segment of the intracellular portion of the mouse CLDN14 and has previously been characterized (25) . The sections were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP, DakoCytomation, Glostrup, Denmark). The DAB + Substrate Chromogen System was used to visualize HRP activity (K3467, DakoCytomation, Glostrup, Denmark). Sections were counterstained with hematoxylin. Light microscopy was carried out using an Olympus BX51 microscope. Animals receiving dihydrotachysterol (DHT; Sigma-Aldrich, St. Louis, MO, USA), a vitamin D analog that is activated by 25-hydroxylation in the liver rather than by 1-hydroxylation in the kidney and renders the animals hypercalcemic, were used as a positive control for CLDN14 expression in the TAL and have been described before (25) .
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