Immunohistochemical (IHC) staining was performed incubating 4 µm FFPE sections in 10 mmol/L citrate buffer (pH 6.0) at 120°C for 5 minutes for antigen retrieval. Endogenous peroxidase was neutralized using EnVision FLEX Peroxidase‐Blocking Reagent (Dako) for 10 minutes. Tissue sections were blocked with 3% bovine serum albumin and incubated with the primary antibodies overnight at 4°C. Primary antibodies used were FRMD6 (Sigma‐Aldrich), ZEB1 (Sigma‐Aldrich), HTR2B (Sigma‐Aldrich), AE1AE3 (Thermo Scientific), CDX2 (Novus Biologicals), SRSF3 (Abcam) and SERPINA1 (Sigma‐Aldrich). After incubation with the EnVision FLEX + mouse or rabbit linker (Dako), EnVision FLEX/HRP (Dako) was used as the secondary antibody for 1 hour at room temperature, followed by 3,3'‐diaminobenzidine (DAB) staining (Dako). CMS molecular classification was performed according to Thrin et al,14 analysing the intensity and content of FRMD6, ZEB1, HTR2B, AE1AE3 and CDX2. SRSF3 and SERPINA1 tumoral epithelial expression was categorized as high (intense staining) and low expression (moderate and negative staining). Individual cores were scored by trained pathologists (CVP and SGL).
Ae1 ae3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AE1/AE3 is a laboratory instrument designed for the detection and analysis of specific proteins. It utilizes immunohistochemical techniques to identify the presence and distribution of target proteins within cells or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Desmin, by Thermo Fisher Scientific (2 mentions)
Alpha smooth muscle actin α sma, by Abcam (2 mentions)
Envision flex peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Srsf3, by Abcam (1 mentions)
Serpina1, by Merck Group (1 mentions)
Envision flex mouse or rabbit linker, by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Vectra polaris, by Akoya Biosciences (1 mentions)
Goat anti mouse igg1 hrp, by Abcam (1 mentions)
Alexa fluor 488 affinipure donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Vim3b4, by Agilent Technologies (1 mentions)
Ll002, by Abcam (1 mentions)
Peroxidase conjugated secondary antibody, by Nichirei Biosciences (1 mentions)
Phalloidin tetramethylrhodamine b isothiocyanate, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Edta buffer, by Merck Group (1 mentions)
11 protocols using ae1 ae3
1
Molecular Subtyping of Cancers via IHC
2
Quantitative Multiplex Immunofluorescence for Tumor Analysis
3
Immunofluorescence Detection of Cytoskeletal Proteins
4
Characterization of Epidermal Equivalents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fixed epidermal equivalents of cultured keratinocytes derived from hESCs and postnatal epidermis by air-lifting culture were routinely processed into 5 μm thick paraffin-wax-embedded sections and haematoxylin and eosin (HE) staining was performed using a standard protocol. Expressions of E-cadherin (CDH1), pan-keratins (KRTs), KRT14, keratin10 (KRT10), IVL, TP63, and vimentin (VIM) were analyzed by immunohistological methods. The sections were treated with mouse monoclonal anti-CDH1 (1:100, 36/E-Cadherin, Becton Dickinson, NJ, USA), mouse monoclonal anti-KRTs (1:100 dilution, AE1/AE3, Thermo Fisher Scientific), mouse monoclonal anti-KRT14 (1:100, LL002, Abcam, Cambridge, UK), rabbit polyclonal anti-KRT10 (1:100, BioLegend, CA, USA), mouse monoclonal anti-IVL (1:1000, SY5, Sigma–Aldrich), mouse monoclonal anti-TP63 (1:100, 4A4, Abcam), and mouse monoclonal anti-VIM (1:100, Vim 3B4, Dako, Agilent Technologies, CA, USA) at room temperature for 90 min and were stained with peroxidase-conjugated secondary antibodies (Nichirei Bioscience, Tokyo, Japan) in accordance with the manufacture's suggested protocol. Human dermal and epidermal tissues were used for negative control of immnohistological analysis, and the results are shown in Supplemental Fig. 2 .
5
Immunostaining of Cytokeratins and Actin Cytoskeleton
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunostaining was performed to confirm the development of cytokeratins (AE1/AE3, 1:250, Thermo Scientific) and the integrity of the actin cytoskeleton (phalloidin-tetramethylrhodamine B isothiocyanate, 1:500, Sigma-Aldrich) in the experiment samples. Immunohistochemistry with the pancytokeratin marker was used to study the development of cytokeratins and urothelial epithelium in urothelial cells as well as after 4 months of in vivo follow-up. The samples were mounted with 4% paraformaldehyde and probed overnight with pancytokeratin primary antibody dilutions. The samples were then treated in a combination of secondary antibody (1:800 Alexa-488 donkey, green fluorescence) and phalloidin the next day. Subsequently, the cell nuclei were dyed with DAPI (1: 2000, Sigma-Aldrich) and examined using a fluorescence microscope (Olympus) [29 ].
6
Urothelial Epithelium Evaluation via Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry with the pancytokeratin marker was used to study the urothelial epithelium after 2, 4 and 16 weeks of follow-up. Briefly, the samples were fixed with 5 % paraformaldehyde and embedded in paraffin. The antigen retrieval was performed by microwaving the samples in 10 mM EDTA buffer (pH 9, Sigma-Aldrich), after which the samples were blocked in 3 % hydrogen peroxide (Sigma-Aldrich). The samples were incubated overnight in diluted primary antibody (1:100, AE1/AE3, Thermo Fisher Scientific, MA, US). On the following day, the secondary antibody (1:200, goat anti-mouse IgG, Thermo Fisher Scientific) was used to detect to the primary antibody.
7
Bladder Construct Perfusability Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After incision of the skin and capsule pouch, the expander was removed, and the bladder construct along with surrounding capsule tissue was exposed. To examine the perfusability of the bladder construct, the ipsilateral femoral artery was separated and injected with 20 ml heparinized Indian ink. Then the construct was harvested and placed in 4% paraformaldehyde. After dehydration and embedding, the tissue was cut into 4 μm‐thick sections for HE staining, immunohistochemistry, and immunofluorescence according to standard staining protocols. Antibodies against AE1/AE3 (1:500; Invitrogen, USA), CD31 (1:500; Novus, USA), desmin (1:100; Invitrogen), alpha‐smooth muscle actin (α‐SMA; 1:500; Abcam, England) and Ki67 (1:500; Abcam) were used for immunohistochemistry. For immunofluorescence, CD31 (1:500; Novus) was applied. In addition, normal bladder tissue was obtained for HE and immunohistochemistry staining.
8
Immunohistochemical Analysis of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the samples were fixed in 4% paraformaldehyde followed by embedding and sectioning. Then PSR staining and immunohistochemical staining were performed according to standard protocols. For immunohistochemical staining, antibodies against AE1/AE3 (1:500; Invitrogen), CD31 (1:500; Novus), desmin (1:100; Invitrogen), alpha‐smooth muscle actin (α‐SMA; 1:500; Abcam), and S100β (1:200; Novus) were applied. In addition, an antibody against desmin (1:100; Invitrogen) was used for immunofluorescence. The results were quantified by Image‐Pro Plus 5.1 software for each tissue sample.
9
Immunostaining of Endometriotic Lesions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen endometriotic lesions were embedded in optimal cutting temperature compound and sectioned at a 5 μm thickness. Then, the sections were washed with PBS, permeabilized with 0.2% Triton X-100, washed with PBS for 10 minutes, and blocked with 10% FBS for 1 hour. The sections were then incubated with primary antibodies overnight at 4°C, washed with PBST, and then, where required, stained with Alexa Fluor–conjugated secondary antibodies for 1 hour at room temperature. The following primary antibodies were used: Goat Anti-human IL-33 (1:40, R&D Systems, AF3625), Mouse Anti-human Vimentin (1:400, Cell Signaling Technology, clone D21H3), Mouse Anti-Pan cytokeratin (1:40, Cell Signaling Technology, clone AE1/AE3), Rabbit Anti-goat Alexa Fluor 647 (1:200; Invitrogen, A27018). Immunostained tissue sections were imaged using the Leica TCS SP8 confocal microscope. Images were taken in a Z-stack (1.20 μm; Z dimension) and tile scan (12 tiles, 580 μm × 435 μm each tile; x and y dimensions) acquisition mode. Two to 3 fields per preparation were imaged using HC PL APO CS2 ×63/1.40 oil objective and LAS-X Software (Leica Microsystems).
10
CTC Isolation and Characterization by Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CTCs are isolated according to their physical properties by Ficoll density gradient centrifugation. In short, whole blood is layered on Ficoll and then centrifuged for 30 min at room temperature. Ery-lysis is done for 3 min and isolated peripheral mononuclear blood cells are cyto-centrifuged onto glass slides (500000 cells per slide)35 (link). For detection of CTCs, immunofluorescence stainings were performed using the pan-cytokeratin antibody (1:80; AE1AE3, eBioScience) and ALDH1 antibody (1:100; ab52492, Abcam) in combination with a species-specific secondary antibody (1:200; anti-rabbit-Alexa488, LifeTechnologies), and a CD45 antibody (1:150; Clone HI30, BioLegend). Cells were fixed with 0.5% para-formaldehyde and blocked with AB serum. All antibodies were incubated for 45 min at room temperature except for ALDH1 (overnight, 4 °C). Glass slides were covered and manually analysed by fluorescence microscopy.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!