Ki-67 staining assay was used to detect the proliferation affected by miR-1306-5p in vivo. It was performed on tumor tissue as a measurement of in situ proliferation. The 4% paraformaldehyde was used for fixing. The 0.1% sodium citrate and 0.1% Triton X were used for permeabilization. The tissues were then sliced into 4 μm sections. The 3% bovine serum albumin/5% goat serum was used for preincubation in PBS for 1 h. Primary antibodies anti-Ki67 (1 : 100, ab15580, Abcam, Cambridge, UK) were added for 1 h. Peroxidase-labelled polymer-conjugated secondary antibodies (1 : 1000, ab214880, Abcam, Cambridge, UK) were added for a 45 min incubation. DAKO Liquid DAB Substrate-Chromogen System was used for 5 min incubation with 3, 3′-diaminobenzidine DAB (DAKO, France). Hematoxylin was used for counterstaining. Then, the sections were dehydrated and coverslipped. The slices were incubated for 1 min by adding 150 μl hematoxylin at a dark room. And then, the slices were washed back to blue and dehydrated and sealed with neutral resin. Images were taken using immunofluorescence microscope (Leica, IX71).
Ab214880
Manufactured by Abcam
Sourced in United Kingdom
Ab214880 is an Anti-CTLA4 antibody. It is a mouse monoclonal antibody that binds to the human CTLA4 protein. This product is intended for laboratory research use only.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Ab16667, by Abcam (2 mentions)
Ab64238, by Abcam (2 mentions)
Ab15580, by Abcam (1 mentions)
Dako liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Dmi3000b, by Leica (1 mentions)
Dab solution, by Solarbio (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Ab125066, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
Ab7481, by Abcam (1 mentions)
Histobond, by Avantor (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab2541, by Abcam (1 mentions)
3 3 diaminobenzidine kit, by Vector Laboratories (1 mentions)
Light microscopy, by Olympus (1 mentions)
Ab8933, by Abcam (1 mentions)
19 protocols using ab214880
1
In Vivo Proliferation Assay of miR-1306-5p
2
Immunohistochemical Analysis of Ki67 in Tumor Samples
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The tumor samples were fixed in 4 % paraformaldehyde, embedded into paraffin and then parceled in 3-µm sections. After deparaffinized, hydrated and blocked treatment (5 % BSA), the tissues were incubated with antibody against Ki67 (ab16667; Abcam) at 4 °C overnight, then added the secondary HRP-conjugated antibody (ab214880; Abcam) and incubated at room temperature for 60 min. DAB solution (DA1015; Solarbio, Beijing, China) was used to produce a dark brown reaction product. Eventually, counterstaining was conducted with hematoxylin reagent (Takara, Beijing, China) and the slides were visualized under a fluorescence microscope (Leica DMI 3000 B, Leica, Germany). Image J software was used for analyzing these images.
3
IHC Detection of ACSL4 and GPX4 in FFPE Tissue
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IHC was performed on formalin-fixed paraffin-embedded tissue sections. ACSL4 and GPX4 were detected using the rabbit anti-ACSL4 antibody (1:100; RRID: AB_2714020; Cat#ab155282, Abcam, UK) and anti-GPX4 antibody (1:100; RRID: AB_10973901; Cat#ab125066, Abcam, UK), respectively [27] (link), [28] (link), [29] (link), [30] (link). Slides were incubated with primary antibodies overnight at 4°C followed by wash and incubation with goat anti-rabbit secondary antibodies (1:1; Cat#ab214880, Abcam, UK) at room temperature for 45 min. Negative controls were treated identically without the primary antibodies.
Biopsy tissue sections were photographed using Leica Aperio CS2 digital pathology scanner (Leica Microsystems Imaging Solutions Ltd, Germany), and analyzed with 4 representative regions of tumor cells randomly captured at ×200 magnification using Leica ImageScope v12.3.2 (Leica Microsystems Imaging Solutions Ltd, Germany). The IHC staining intensity of ACSL4 and GPX4 was analyzed by Image-Pro Plus v6.0 (Media Cybernetics Inc, USA). Protein expression was presented by mean optical density, which was calculated as the integrated optical density (IOD) of positively stained area divided by tumor area in each image [31] .
Biopsy tissue sections were photographed using Leica Aperio CS2 digital pathology scanner (Leica Microsystems Imaging Solutions Ltd, Germany), and analyzed with 4 representative regions of tumor cells randomly captured at ×200 magnification using Leica ImageScope v12.3.2 (Leica Microsystems Imaging Solutions Ltd, Germany). The IHC staining intensity of ACSL4 and GPX4 was analyzed by Image-Pro Plus v6.0 (Media Cybernetics Inc, USA). Protein expression was presented by mean optical density, which was calculated as the integrated optical density (IOD) of positively stained area divided by tumor area in each image [31] .
4
Immunohistochemical Analysis of Biological Samples
5
Immunohistochemical Analysis of Synovial Tissue
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Paraffin-embedded synovial tissue sections were dewaxed through an ethanol gradient and incubated with 3% hydrogen peroxide in methanol for 10 min at room temperature to quench endogenous peroxidases and then boiled in 0.01 M sodium citrate buffer (pH = 6) for antigen retrieval, washed with PBS, and blocked with 10% normal goat serum (Thermo Fisher Scientific, USA) for 20 min at 37°C. The sections were incubated overnight with primary antibodies against XIAP (ab2541, Abcam, UK), Bcl-2 (ab59348, Abcam, UK), and Bax (ab32503, Abcam, UK) at 4°C and then incubated with the goat anti-rabbit IgG H&L (HRP polymer) (ab 214880, Abcam, UK) secondary antibody for 30 min at 37°C. After immersing in DAB (Wuhan Bioswamp Biological Co., Ltd., China) for coloration, the sections were rinsed with distilled water, counterstained with hematoxylin, dehydrated, and mounted. The stained sections were observed under a microscope (Olympus Corporation, Japan). The integrated optical density (IOD) was quantified using Image-ProPlus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and the mean optical density (MOD) of each sample was calculated.
6
Immunohistochemical Analysis of PI3K/AKT Pathway
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The method of immunohistochemistry was performed as previously described (18 (link)). The liver tissues of rats were fixed using 4% paraformaldehyde for 30 min at 25°C, and then dehydrated, embedded in paraffin and sliced into sections of 4 µm thickness. Sections were deparaffinized with xylene and were incubated with primary antibodies against phosphorylated (p)-PI3K (1:500; cat. no. ab182651; Abcam, Cambridge, MA, USA), PI3K (1:500; cat. no. ab135253; Abcam), p-Thr308-AKT (p-AKT; 1:500; cat. no. ab8933; Abcam), and AKT (1:500; cat. no. ab8805; Abcam) overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. nos. ab214880 and ab97040; Abcam) for 2 h at 25°C. Then, the sections were incubated using a 3,3′-diaminobenzidine kit (Vector Laboratories, Inc.) according to the manufacturer's protocols. The expression of each protein was observed by light microscopy (Olympus Corporation); five fields in each image were analyzed. The findings were analyzed determined using Image Pro Plus 6.0.
7
Immunohistochemical Analysis of Intestinal Tight Junction Proteins
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The ileal tissue specimens were cut into 10 µm sections following dewaxing and hydrating. Sections were treated with 3% H2O2-methanol to block endogenous peroxidase activity, following which they were incubated with 5% normal goat serum (Wuhan Boster Biological Technology, Ltd.) at room temperature for 10 min and incubated with ZO-1 and occludin antibodies (dilution 1:200) overnight at 4°C. Slides were subsequently washed with PBS and incubated with a biotinylated secondary antibody (1:500; cat. no. SA00004-2; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature. Slides were washed with PBS again and incubated with HRP-labeled streptavidin (1:200; ab214880; Abcam) at 37°C for 1 h. Samples were developed using diaminobenzene (DAB) at room temperature for 30 sec and counterstained with hematoxylin at room temperature for 5 min. Slides were rinsed in distilled water and dehydrated, following which they were observed under a light microscope (magnification, ×100).
8
Protein Expression Analysis using SDS-PAGE and Western Blotting
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The harvested cells were lysed using RIPA buffer (150 mM NaCl, 1% NP-40, 1 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl, pH 7.4, 0.25% sodium deoxycholate) and protease inhibitor cocktail (Roche Diagnostics, Mannheim, German). The protein concentration was measured using a BCA protein assay kit (Pierce, Waltham, MA, USA) according to manufacturer’s protocol. Equal amounts of protein were separated electrophoretically on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Roche, Basel, Switzerland). This was followed by blocking with 5% skim milk at room temperature for 1 h; the membranes were incubated with indicated antibodies at 4°C overnight. After washing with Phosphate Buffered Solution Tween-20 three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated second antibodies at room temperature for 1 h. Finally, the blots were visualized using enhanced chemiluminescence reagent (Pierce, Minneapolis, MN, USA) according to manufacturer’s instructions. The antibodies used were as follows: FOXA1 (1:1000; Abcam, Cambridge, UK; ab23738), cyclin D1 (1:3000; Abcam; ab134175), β-actin (1:5000; Abcam; ab8227), HRP-conjugate goat anti-mouse (1:5000; Abcam; ab214879) and HRP-conjugate goat anti-rabbit (1:5000; Abcam; ab214880).
9
Evaluating Synovitis and Osteoclasts in Hindlimb Injury
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Whole hindlimbs were resected from each animal and placed in 10% neutral buffered formalin for fixation. Hindlimbs were subsequently rinsed in saline and decalcified with Cal-EX™ solution (Catalog # CS510-1D, ThermoFisher) for 14 days. Decalcified samples were embedded in paraffin blocks for fixation and sectioning. Coronal sections were cut to a thickness of five microns and stained with hematoxylin and eosin (H&E) for histologic analysis. To evaluate synovitis, H&E sections were used to evaluate vascularity (0–2), Detritus (0–2) and Fibrosis (0–3) as previously described [15 (link)]. In addition to H&E, sections were stained for presence of osteoclasts (OC) at Day 14 and Day 56 post-injury using the OC surface marker DC-STAMP. Briefly, decalcified and rehydrated samples were blocked with 5% normal goat serum (NGS) for 1 h then incubated with DC-STAMP (Catalog # ab238151, Abcam) diluted 1:50 with NGS. Following incubation, slides were incubated in goat-anti-rabbit-HRP secondary (Catalog # ab214880, Abcam) and then developed with DAB substrate (Catalog # SK-4105, Vector labs).
10
Histological Assessment of Liver Fibrosis
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FFPE liver sections (2 to ~3 μm) were stained with H&E to visualize morphology or Sirius Red (catalog 196-16201; Wako) to assess collagen deposition. For F4/80 detection, liver sample slides were reacted with an F4/80 Ab (1:200 dilution; ab111101; Abcam; RRID:AB_10859466) at 4°C overnight followed by a secondary HRP polymer-conjugated goat anti–rabbit IgG Ab (1:2,000 dilution; ab214880; Abcam) for 60 minutes at room temperature and were developed with DAB (Dako). After counterstaining with hematoxylin, representative images were acquired using DP2-BSW software (Olympus).
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