Tumor samples were distributed in 15 TMAs using 4 mm tissue cores at the Experimental Pathology Laboratory of Pontifical Catholic University of Paraná (PUCPR). From each donor block was extracted one or two cylinders 4 mm in diameter and deposited in the receiver blocks, previously prepared. In each TMA, a sample of normal breast tissue was included as an internal control. Thereafter, 4 μm tissue sections from the TMA blocks were transferred to electrically charged Star Frost® (Braunschweig, Germany) slides and incubated with primary antibodies [anti-CD44 (anti-human mouse monoclonal, clone DF1485, dilution 1/40, Novocastra, Newcastle, UK), anti-CD44v6 (antihuman mouse monoclonal, clone VFF-7, dilution 1/100, Novocastra, Newcastle, UK), anti-CD24 (anti-human rabbit polyclonal, dilution 1/200, Abbiotec, San Diego, CA, USA), and anti-ALDH1 (anti-human rabbit monoclonal, clone E-P1932y, dilution 1/100, Epitomics, Cambridge, Massachusetts, USA)] for 12 h in a humidified chamber at 2−8°C. An Advance Dako (Caripenteria, CA, USA) secondary antibody was incubated with the slides for 30 min at 2−8°C. The reactions were developed using a DAB chromogen-substrate solution (Dako). Harris hematoxylin was used for counterstaining. Positive and negative (incubated without primary antibody) controls were run in parallel with all reactions.
Dab substrate chromogen solution
Manufactured by Agilent Technologies
Sourced in United States
The DAB substrate-chromogen solution is a laboratory reagent used in immunohistochemistry and related techniques. It provides a brown-colored signal for the visualization of target antigens or proteins in tissue samples. The solution contains 3,3'-Diaminobenzidine (DAB) and the necessary cofactors for the chromogenic reaction. When applied to prepared slides, the DAB substrate-chromogen solution reacts with the enzyme label, resulting in a visible brown precipitate at the site of the target.
Automatically generated - may contain errors
Lab products found in correlation
Tissuequest, by TissueGnostics (2 mentions)
Envision mouse rabbit peroxidase detection system, by Agilent Technologies (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Anti cd44, by Leica (1 mentions)
Df1485, by Leica (1 mentions)
E p1932y, by Abcam (1 mentions)
Biotinylated chain specific anti human immunoglobulins, by Southern Biotech (1 mentions)
Streptavidin horseradish peroxidase complex, by Southern Biotech (1 mentions)
Citrate buffer solution, by Merck Group (1 mentions)
Ab5690, by Abcam (1 mentions)
Envision dual link system hrp dab kit, by Agilent Technologies (1 mentions)
Ncl l ki67 mm1, by Leica (1 mentions)
Ab152, by Merck Group (1 mentions)
Nef812001ea, by PerkinElmer (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti khdrbs3 slm 2 f 3 antibody, by Santa Cruz Biotechnology (1 mentions)
Ab109263, by Abcam (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Lsab2 streptavidin biotin complex system, by Agilent Technologies (1 mentions)
17 protocols using dab substrate chromogen solution
1
Immunohistochemical Profiling of Breast Tumors
2
Immunohistochemical Analysis of QSOX1 in Brain Tumors
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Histological slides stained with hematoxylin-eosin were reviewed, and the samples classified according to their histological type. Two representative samples of each tumor (fifty-eight cores) and twelve samples/cores of non-neoplastic cerebellum were selected and used to prepare five tissue microarrays (TMAs) [5 (link)].
Histological sections were cut from these blocks and incubated with mouse anti-human-QSOX1 recombinant monoclonal antibody produced at the Federal University of Paraná (UFPR) [3 (link),6 (link)]. AdvanceTM HRP Dako® was used as the secondary antibody. The immune reactions were developed by adding DAB chromogen-substrate solution (Dako®) to the slides.
Histological sections were cut from these blocks and incubated with mouse anti-human-QSOX1 recombinant monoclonal antibody produced at the Federal University of Paraná (UFPR) [3 (link),6 (link)]. AdvanceTM HRP Dako® was used as the secondary antibody. The immune reactions were developed by adding DAB chromogen-substrate solution (Dako®) to the slides.
3
Immunohistochemical Staining of Rodent Tissues
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Portions of Sprague–Dawley rat sural nerves or NXS2 tumors grown in A/J mice were embedded in Tissue Tek-II O.C.T. (Miles, Naperville, IL), snap frozen in liquid nitrogen, and stored at −80°C. Ten micrometer-sections were cut, fixed in acetone, and stained with chimeric mAbs c.8B6 or ch14.18 for 1 hour. After washing with PBS, mAb binding was detected by stepwise incubation with biotinylated chain specific anti-human immunoglobulins (1∶500, diluted) (Southern Biotech, Birmingham, AL, USA) for 1 hour at room temperature, followed by streptavidin-horseradish peroxidase complex (1∶1000, diluted) (Southern Biotech, Birmingham, AL, USA) for 1 hour at room temperature. After rinsing, the bound antibody was detected with DAB chromogen substrate solution (Dako, Glostrup, Denmark), which was used to produce a brown deposit. Rituximab was used as a negative control. A mAb specific to mouse neural cell adhesion molecule (CD56) purchased from Abbiotec (San Diego, CA, USA) was used as positive control. The concentration 5 µg/mL was selected for the study because it would result in minimal background and maximal detection signal. Slides were counter-stained with hematoxylin before immunocytological evaluation. Staining was graded as positive or negative according to the presence or absence of immunoreactivity, respectively.
4
Immunohistochemical Detection of T and B Lymphocytes
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The Dako Envision® + Dual Link System-HRP (DAB+) kit (Dako K4965, USA) was used, with a slight modification of the protocol, to detect apoptotic cells in the spleen. Tissue sections, prepared as above, were deparaffinized, rehydrated with ascending concentrations of ethanol (100, 90, and 70%), and washed in distilled water. Wax-enclosed sections were then flooded with dual endogenous enzyme (Dako K4065, USA) as a blocking agent and incubated for 10 min. Sections were then washed with citrate buffer solution (10 mM, pH 6.0) (Sigma, USA), immersed in tris-buffered saline with Tween-20 (TBST) for 3 min, and then incubated with CD3 primary antibody (T-lymphocyte marker) (Abcam ab5690, UK) or CD19 primary antibody (B-lymphocyte marker) (Bioss bs0079R, USA) at 4°C overnight. After washing with TBST, sections were incubated for 45 min with labeled polymer-HRP reagent (Dako K4065, USA) and then washed again with TBST. DAB + substrate-chromogen solution (Dako K4065, USA) was then applied for 3 min. Finally, sections were counterstained with Myer's hematoxylin, mounted in DPX medium, and observed under a light microscope at 40x magnification.
5
Immunohistochemical Analysis of TDO2 and Other Markers
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Sections of 3 μm thickness were used for immunohistochemical analysis. The immunohistochemical staining procedures were performed as previously described [12 (link)]. The expression of TDO2 was scored as described in the previous study, which combined the intensity (1+, 2+, 3+) and the percentage (from 0 to 100%) of tumor cells expressing TDO2 [12 (link)]. Immunohistochemical staining of ALDH1, CD44, CD133, EGFR, HER2, p53, and PDL1 was performed as previously described [18 ]. Primary antibodies and dilution ratios were described in detail in Table S1 . The sections were incubated with primary antibody for 1 h at room temperature, followed by incubation with Dako Envision+ Peroxidase Detection System (Dako Cytomation, Carpinteria, CA, USA) (Anti-mouse or Anti-rabbit) 1 h at room temperature. The sections were incubated with the DAB Substrate-Chromogen Solution (Dako Cytomation) for 10 min for the color reaction. Two surgical pathologists (D.T. and K.S.) independently examined the immunohistochemical results without knowledge of clinicopathologic information or patient outcome.
6
Immunohistochemical Analysis of Mucins and Markers
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Immunohistochemical analysis was performed with a Dako Envision + Mouse/Rabbit Peroxidase Detection System (Dako Cytomation, Carpinteria, CA, USA). Antigen retrieval was performed using a pressure cooker heated to 100 °C in citrate buffer (pH 6.0) for 5 min. Peroxidase activity was blocked with 3% H2O2-methanol for 10 min.
Sections were incubated with anti-MUC5AC (anti-MRQ-19, 760-4389, V0001348, Roche; 1:2 dilution for IHC), anti-MUC6 (NCL-MUC-6, 6014968, Leica Biosystems; 1:100 dilution for IHC), anti-MUC2 (anti-MRQ-18, 760-4388, V0001436, Roche; 1:2 dilution for IHC), anti-CD10 (56C6, CD10-270, 602420, Leica Biosystems; 1:100 dilution for IHC), or anti-Ki67 (NCL-L-Ki67-MM1, Leica Biosystems; 1:100 dilution for IHC) antibodies for 1 h at 25 °C, followed by incubation with Envision and the anti-mouse peroxidase for 1 h at room temperature. For color reactions, sections were incubated with DAB substrate-chromogen solution (Dako Cytomation) for 5 min. Sections were counterstained with 0.1% hematoxylin & eosin. Reactions lacking a primary antibody were used as negative controls.
Sections were incubated with anti-MUC5AC (anti-MRQ-19, 760-4389, V0001348, Roche; 1:2 dilution for IHC), anti-MUC6 (NCL-MUC-6, 6014968, Leica Biosystems; 1:100 dilution for IHC), anti-MUC2 (anti-MRQ-18, 760-4388, V0001436, Roche; 1:2 dilution for IHC), anti-CD10 (56C6, CD10-270, 602420, Leica Biosystems; 1:100 dilution for IHC), or anti-Ki67 (NCL-L-Ki67-MM1, Leica Biosystems; 1:100 dilution for IHC) antibodies for 1 h at 25 °C, followed by incubation with Envision and the anti-mouse peroxidase for 1 h at room temperature. For color reactions, sections were incubated with DAB substrate-chromogen solution (Dako Cytomation) for 5 min. Sections were counterstained with 0.1% hematoxylin & eosin. Reactions lacking a primary antibody were used as negative controls.
7
Quantifying Dopaminergic Neuron Density
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TH immunostaining was used to demonstrate the distribution of dopaminergic neurons. Sections from the SN and striatum were selected. Free-floating brain sections were incubated overnight at 4 °C in rabbit anti-TH antibody (1:200, AB152, EMD Millipore, Burlington, MA, USA), followed by 1 h in biotinylated goat anti-rabbit secondary antibody (1:500, NEF812001EA, PerkinElmer, Waltham MA, USA). Immunoreactivity was visualized by incubating the sections in a DAB Substrate-Chromogen solution (DAB; K3468, Dako Cytomation) for 3 min. The sections were washed three times with PBS and then mounted onto gelatin-coated slides. The number of TH-positive cells in the SNpc was counted while using a light microscope (LSM510; Carl Zeiss, Jena, Germany). TH-immunoreactive fiber density in the striatum was measured using TissueFAXS and analyzed while using TissueQuest (Tissue Gnostics, Vienna, Austria).
8
Immunohistochemical Analysis of Neuroinflammation
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Microglial cells and astrogliosis in the SN and striatum were assessed using Iba-1, CD68, and GFAP immunostaining. Endogenous peroxidase activity was neutralized by a 20 min. incubation in 0.3% H2O2. After washes in PBS containing 0.05% Triton-X (dilution media), background staining was blocked by a 1 h incubation in a Tris buffered saline solution containing 3% normal horse serum, 2% bovine serum albumin, and 0.05% Triton X-100. The sections were then incubated with the required primary antibody: anti-GFAP (Millipore, 1: 1000), anti-Iba1 (Chemicon, 1:500), or anti-CD68 (Chemicon, 1:300). After washes in dilution media, the sections were placed in the avidin biotin (Elite ABC kit, Vector Laboratories, Burlingame, CA) substrate (1:1000) for 75 min. The sections were then washed in 0.1 M imidazole/1.0 M acetate buffer, pH 7.4, and allowed to react in a DAB Substrate-Chromogen solution (K3468, DAKO, Carpinteria, USA). Nickel sulfate was added to the DAB chromogen reaction. Immunoreactivity was measured while using TissueFAXS and analyzed using TissueQuest (Tissue Gnostics, Vienna, Austria).
9
Immunohistochemical Analysis of KHDRBS3
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Immunohistochemical analysis was performed with a Dako Envision+Mouse/Rabbit Peroxidase Detection System (Dako Cytomation). Antigen retrieval was performed by pressure cooker heating in citrate buffer (pH 6.0) for 5 min. Peroxidase activity was blocked with 3% H2O2‐methanol for 10 min. Sections were incubated with a mouse monoclonal anti‐KHDRBS3 (SLM‐2) F‐3 antibody (1:200; Santa Cruz Biotechnology, Inc) for 1 h at room temperature, followed by incubation with Envision+anti‐mouse peroxidase for 1 h. For color reactions, sections were incubated with DAB Substrate‐Chromogen Solution (Dako Cytomation) for 5 min. Sections were counterstained with 0.1% hematoxylin. Reactions lacking a primary antibody were used as negative controls.
Two surgical pathologists (NS and DT) independently measured the ratio of positivity without knowledge of the clinical and pathological parameters or outcome of the patients. When >50% of tumor cells were stained, immunostaining was considered to be positive for KHDRBS3 in reference to the median value of the positivity. Inter‐observer differences were resolved by consensus review at a double‐headed microscope after independent reviews.
Two surgical pathologists (NS and DT) independently measured the ratio of positivity without knowledge of the clinical and pathological parameters or outcome of the patients. When >50% of tumor cells were stained, immunostaining was considered to be positive for KHDRBS3 in reference to the median value of the positivity. Inter‐observer differences were resolved by consensus review at a double‐headed microscope after independent reviews.
10
Immunohistochemical Staining of Laryngeal Lesions
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Specimens of laryngeal carcinomas and benign lesions were removed and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4 °C, embedded in paraffin and sectioned at 4 μm. Deparaffiisation, hydration and epitope demasking were carried out with a microwave antigen retrieval procedure in sodium citrate buffer for 5 min. Then sections were treated with 3% H2O2 for 20 min to quench endogenous peroxidase activity and blocked with 5% bull serum albumin (BSA) for 20 min. The primary antibody used were goat anti-rabbit anti-CCTα (1:50, ab109263, Abcam, Cambridge, UK). Slides stained with primary antibodies were incubated at 4 °C overnight. Then the slides were incubated with an anti-rabbit IgG antibody at room temperature for 30 minutes. The binding of the primary antibody to the sections was visualized by using DAB Substrate-Chromogen Solution (Dako Cytomation, Carpinteria, CA, USA), the sections were then counterstained with hematoxylin (Beyotime Biotechnology).
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