The level of UBC9 in paraffin-embedded tissue sections was detected with immunohistochemical staining. A goat monoclonal antibody was used as the primary antibody. Paraffin-embedded tissues were pretreated at 65°C for 2 h, followed by deparaffinization using standard procedures. Antigen retrieval was carried out with an antigen retrieval solution (10 mmol/L Tris, 1 mmol/L EDTA, Ph 9.0) before 2% sheep serum was added. Then, the slides were incubated with the antibody for UBC9 (ab21193, Abcam) (1:200 dilution) at 4°C overnight. Next, the slides were labeled with EnVision HRP kits (DAKO) at room temperature for 30 minutes, incubated with DAB substrate liquid (DAKO), and counterstained with Mayer› s hematoxylin(DAKO). All of the sections were observed and photographed with a light microscope using a DP70 CCD system (Olympus Corp.).
Liquid dab substrate
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Liquid DAB Substrate is a chromogenic substrate used in immunohistochemistry and immunocytochemistry applications. It produces a brown, water-insoluble precipitate upon reaction with the enzyme used to label the target antigen. This substrate provides a visible signal to indicate the presence and localization of the target protein in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Mayer s hematoxylin, by Agilent Technologies (2 mentions)
Envision hrp kit, by Agilent Technologies (1 mentions)
Dp70 ccd system, by Olympus (1 mentions)
Amersham ecl detection kit, by GE Healthcare (1 mentions)
Calf serum, by Lonza (1 mentions)
Anti rabbit hrp conjugated secondary antibody, by Merck Group (1 mentions)
Trichrome masson staining reagents, by Merck Group (1 mentions)
Fitc conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Hematoxylin eosin, by Merck Group (1 mentions)
Evans blue, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Envision system hrp labeled polymer, by Agilent Technologies (1 mentions)
Leica scn 400f scanner, by Leica camera (1 mentions)
Anti brdu primary antibody, by BD (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Axio imager a1, by Zeiss (1 mentions)
Ab203615, by Abcam (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Ab97508, by Abcam (1 mentions)
12 protocols using liquid dab substrate
1
Immunohistochemical Detection of UBC9 in Paraffin Sections
2
Immunohistochemical Analysis of WASF2 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were deparaffinized, rehydrated, and microwave‐heated in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval. The sections were then incubated with 0.3% hydrogen peroxide and non‐specific binding was blocked using BSA. For incubation with primary mAb, tissue slides were incubated at 4°C overnight with rabbit anti‐WASF2 mAb (1:1000; Cell Signaling Technology, Danvers, MA, USA). They were then labeled by EnVision HRP (goat) kits at room temperature for 30 min, incubated with DAB substrate liquid (Dako, Glostrup, Denmark), and counterstained by Mayer's hematoxylin (Dako). Intensity of staining was graded on a three‐point scale (intensity scores): 0, negative; 1, weak; and 2, strong. The percentage of positive cells was divided into five grades (percentage cores): 0, 0%; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The histological score (H score) was determined by the following formula: overall score = intensity score × percentage score. An overall score of 0–8 was calculated and graded as: score 0, 0; score 1, 1–3; score 2, 4–6; or score 3, 7–8.
3
Transcardial Perfusion and Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were anesthetized with isoflurane inhalation, and blood was flushed with phosphate buffered saline (PBS) by transcardial perfusion. The patch and IVC were explanted and fixed with 4% PFA (15 min) prior to incubation with primary antibodies overnight at 4 °C. The patch and IVC were then incubated with EnVision reagents for 1 h at room temperature and treated with Dako Liquid DAB Substrate. Samples were directly visualized with a dissecting microscope under 63× magnification.
4
Cardiac Tissue Staining and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
General cell culture supplies were purchased from BD Biosciences (Spain); calf serum was from BioWhittaker (Verviers, Belgium). Cell culture-grade gelatin, trypsin, antibiotics, hematoxylin-eosin, Trichrome Masson staining reagents, Evans blue, and TTC dyes were from Sigma (Spain). HRP-conjugated anti-mouse secondary antibody and liquid DAB substrate were from Dako (Carpinteria, CA). Anti-MMP-2, MMP-9, CD68, and FITC-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-heavy chain cardiac myosin antibody was from Abcam (Abcam, UK). HRP-conjugated anti-rabbit secondary antibody was from Sigma-Aldrich. Amersham ECL detection kit was from GE (GE Healthcare Life Sciences, Spain). Centrifugation concentrators were from Sartorius (Fischer Scientific, Spain).
5
Quantifying KPC Tumor Cell Invasion
6
Immunohistochemical Detection of Phosphorylated CD19
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleen sections were stained for phosphorylated CD19 using an anti-CD19 (phospho Y531) antibody (1:200) by Abcam (ab203615) according to the manufacturers' instructions. Rabbit polyclonal IgG-ChIP Grade (ab27478) was used as isotype control. Biotinylated goat anti-rabbit IgG (H+L) by Vector (BA-1000) was used as a secondary antibody, followed by incubation with streptavidin-peroxidase polymer (Sigma-Aldrich, S2438) and detection with Liquid DAB Substrate (DAKO K3466). Hematoxylin (Mayer's hematoxylin; Applichem, APC2-254766.1611) was applied as counterstain. Images were taken with Axio Imager A1 from Zeiss.
7
Immunohistochemical Assessment of DAXX, ATRX, and MEN1 Protein Expression in pNEN
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression of the DAXX, ATRX, and MEN1 genes, which is altered by most mutations, was determined by IHC performed on pNEN tissue microarrays (TMA; refs. 25, 26 (link)). Following surgical resection, tumor specimens were fixed, embedded in paraffin and TMAs generated and sectioned by the UAB Pathology Core Research Lab. Slides were rehydrated using xylene and ethanol. Antigen retrieval was accomplished by immersing slides in citrate buffer (pH 6) and placing them in a pressure cooker for 10 minutes. Antibodies to Daxx (Sigma, HPA008736), Atrx (Abcam, ab97508), and Menin (Abcam, ab92443) were diluted at a 1:200, 1:700, and 1:100, respectively, in PBS augmented with 0.3% Tween 20 and 5% goat serum. TMA sections were incubated in primary antibodies overnight at 4°C. Following biotin and peroxidase blocking, sections were incubated with an anti-rabbit biotin–labeled secondary antibody (Pierce goat anti-rabbit IgG, #31820) for 1 hour at room temperature. Slides were then stained with DAB chromogen (Dako Liquid DAB+ substrate) and counterstained with hematoxylin. TMA stains were then evaluated in a blinded manner by a board-certified pathologist specializing in GEP-NENs. All studies of patient-derived tissues were approved by the University of Alabama at Birmingham Institutional Review Board (IRB-300006067).
8
Immunohistochemical Detection of Fibrinolytic Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histological sections (3 μm) were obtained from the paraffin-embedded specimens and mounted on glass slides with organosilane adhesive (3-aminopropyltriethoxysilane; Sigma Chemical Co., St. Louis, USA). For deparaffinization, rehydration, and antigen retrieval, the sections were immersed in Trilogy solution (1:100; Cell Marque, Rocklin, USA) in a Pascal pressure cooker. After these steps, endogenous peroxidase was blocked by treatment with 3% hydrogen peroxide. The sections were incubated overnight in a moist chamber with the following primary antibodies: anti-uPA (sc-14019, 1:800, Santa Cruz Biotechnology, Dallas, USA), anti-uPAR (E-3, 1:200, Santa Cruz Biotechnology, Dallas, USA), and anti-PAI-1 (C-9, 1:400, Santa Cruz Biotechnology, Dallas, USA). The sections were then rinsed twice in phosphate-buffered saline (PBS) and incubated with the HiDef Detection HRP Polymer System (Cell Marque, Rocklin, USA) at room temperature. The reactions were developed using diaminobenzidine (Liquid DAB + Substrate; Dako, Carpinteria, USA) as chromogen. Finally, the sections were counterstained with Mayer's hematoxylin and coverslipped. Endothelial cells present in the histological sections were used as positive internal control. Sections in which the primary antibodies were omitted served as negative control.
9
Immunohistochemical Detection of IFNε and FoxP3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect IFNε in human colon samples, heat-induced epitope retrieval (HIER) was performed with 10 mmol/L Trisma acetate and 1 mmol/L EDTA buffer, pH 6.0 in a microwave. IFNε was detected using rabbit anti-IFNε (Prestige Antibodies), and background staining was prevented with CAS block (Invitrogen). Biotinylated anti-rabbit immunoglobulin G (Vector Laboratories) was used as secondary antibody. To stain FoxP3-expressing cells in mouse colon sections, HIER was done using a Biocare decloaking chamber (Biocare) in Tris-EDTA Buffer (10 mmol/L Tris Base, 1 mmol/L EDTA solution, 0.05% Tween 20, pH 9.0) at 110°C for 5 minutes under pressure. FoxP3 was detected with rat anti-mouse FoxP3 (eBioscience). Biotinylated anti-rat immunoglobulin G (Vector Laboratories) was used as secondary antibody. This was followed by incubation with VECTASTAIN Elite ABC-HRP (Vector Laboratories). Color development was performed with liquid DAB+ Substrate (Dako), and slides were counterstained with hematoxylin (Invitrogen) and coverslipped with D.P.X. neutral mounting medium.
Slides were scanned at 20× magnification with an Aperio Scanscope AT Turbo (Leica Biosystems), and intensity of staining was analyzed with Aperio ImageScope software (Leica Biosystems).
Slides were scanned at 20× magnification with an Aperio Scanscope AT Turbo (Leica Biosystems), and intensity of staining was analyzed with Aperio ImageScope software (Leica Biosystems).
10
Quantifying CDK1 Expression in Breast Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue microarray (TMA) sections containing both normal and cancerous breast tissues were retrieved from Cooperative Human Tissue Network, the University of Virginia. TMAs were deparaffinized and rehydrated using standard methods. The sections were then sequentially incubated with rabbit polyclonal anti-CDK1 (Sigma, St. Louis, MO), biotin-conjugated goat anti-rabbit, and ExtrAvidin Peroxidase (ExtrAvidin Kit, Sigma). Staining was developed with liquid DAB substrate (DAKO, Carpinteria, CA), sections were counterstained with hematoxylin, and mounted with Permount (Fisher Scientific, Pittsburgh, PA). Stained TMAs were scanned, and digital images were obtained with Aperio Scanscope System (Leica Biosystems, Vista CA). The intensity of CDK1 staining in a tissue from each patient was evaluated with the Positive Pixel Count Algorithm (Leica Biosystems). This algorithm quantifies the amount of specific stain present in a digital slide by evaluating an average intensity of all pixels for subsequent calculation of the optical density and the proportion of positively stained area.
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!