Parafin-embedded tissues were cut into 5 μm-thick sections, deparaffinized in xylene, and rehydrated in graded ethanol. Sections were incubated with anti-CD68 antibodies overnight at 4 °C and then incubated with SignalStain®Boost IHC Detection Reagent (HRP, Rabbit) as indicated by the manufacturer (Cell Signaling Technology, Danvers, MA, USA) for 30 min at room temperature. Finally, the sections were stained with DAB + Liquid according to manufacturer’s protocol (DAKO, Glostrup, Denmark).
Liquid dab
Manufactured by Agilent Technologies
Sourced in United States, Denmark
Liquid DAB is a chromogenic substrate used for immunohistochemical (IHC) staining in microscopy applications. It provides a brown colorimetric reaction when catalyzed by horseradish peroxidase (HRP) enzyme labels, allowing visualization of target molecules in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Pt link, by Agilent Technologies (3 mentions)
Eclipse 400, by Nikon (3 mentions)
Histofine mouse max po anti rat and anti rabbit, by Nichirei Biosciences (2 mentions)
Evolution vr cooled color 13 bit digital camera, by Media Cybernetics (2 mentions)
Advance tm hrp, by Agilent Technologies (2 mentions)
Signalstain boost ihc detection reagent, by Cell Signaling Technology (1 mentions)
Normal goat igg, by R&D Systems (1 mentions)
Dako real antibody diluent, by Agilent Technologies (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Mildform 10n, by Fujifilm (1 mentions)
Ab 108 c, by R&D Systems (1 mentions)
Envision flex hematoxylin, by Agilent Technologies (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Biotinylated peanut agglutinin pna, by Vector Laboratories (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Envision system hrp labelled polymer anti mouse, by Agilent Technologies (1 mentions)
Ab66155, by Abcam (1 mentions)
Envision hrp, by Agilent Technologies (1 mentions)
H 142, by Santa Cruz Biotechnology (1 mentions)
33 protocols using liquid dab
1
CD68 Immunohistochemistry Protocol
2
IHC analysis of EPHA2 in breast and gastric tumor xenografts
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Female nude mice bearing MDA-MB-231 and SNU-16 tumors were euthanized by cervical dislocation under anesthesia, and subcutaneous tumors were dissected. After dissection, tumor tissues were fixed with 10% neutral buffed formalin (Mildform®10N, Wako Pure Chemicals, 133-10311) for 2 days, and processed for standard formalin-fixed paraffin-embedded sections. IHC was performed using Autostainer Link 48 (DAKO) and PT Link (DAKO) at room temperature, unless otherwise stated. All of the primary antibodies (Goat anti-EPHA2 Ab [R&D Systems, AF3035] and Normal goat IgG [R&D Systems, AB-108-C]) were diluted to a final concentration of 2.5 μg/mL with the DAKO REAL Antibody Diluent (DAKO, S2022). Approximately 4 μm thick sections were deparaffinized and pretreated with Envision FLEX TRS High (DAKO, K8004) for 40 min at 97°C followed by endogenous peroxidase blocking (DAKO, S2023) and protein blocking (DAKO, X0909). Then, the sections were sequentially incubated with the primary antibodies for 1 h, Histofine Simple Stain MAX-PO (Goat) (Nichirei Bioscience, 414161) as a secondary antibody for 30 min, and DAB+ Liquid (DAKO, K3468) for 10 min. Washes were performed after every step. Finally, the sections were counterstained with EnVision FLEX Hematoxylin (DAKO, K8008) for 5 min, and mounted. Two individual tumors from each xenograft model were used for IHC.
3
Vascular Graft Engraftment Dynamics
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The early stage of the engraftment of the vascular grafts was analyzed 10 days (n = 3), and the long-term response was assessed 6 months (n = 4) following implantation. The tissue blocks were cut so as to provide approx. 10 µm-thick histological sections with a section plane parallel to the long axis of the aorta (Figure 1 ).
The sections were stained using a variety of general and specialized histological (Table 2 ) as well as immunohistochemical reaction dyes (Table 3 ) in order to enable the characterization of the cellular populations of the grafts. The visualization of the immunohistochemical reaction was based on diaminobenzidine (DAB+, Liquid; DakoCytomation, Glostrup, Denmark). The immunohistochemical sections were then counterstained with Gill’s hematoxylin. All the sections were dehydrated in graded ethanol solutions and mounted with a xylene-soluble medium.
Stained samples were evaluated qualitatively in order to describe the vessel remodeling in two investigated time points. No statistical analysis was performed due to the low number of tested samples.
The sections were stained using a variety of general and specialized histological (
Stained samples were evaluated qualitatively in order to describe the vessel remodeling in two investigated time points. No statistical analysis was performed due to the low number of tested samples.
4
Quantitative Analysis of LAMC1 Expression in Glioma
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A tissue microarray of glioma (Cat: HBraG159Su01) and normal brain tissue sections were purchased from Shanghai Outdo Biotech (Shanghai, China). IHC was performed using a DAKO automatic IHC instrument in accordance with the manufacturer’s protocol. The array was incubated with an anti-LAMC1 antibody (Cat#: ABP55085, Abbkine, Wuhan, China) at a 1:100 dilution overnight at 4 °C and developed using Dako Liquid DAB. The scoring system of the staining intensity was as follows: negative (0 points); weak (1 points); moderate (2 points); strong (3 points). The positive range percentage criteria were as follows: 0%–25% positive cells (1 points); 26%–50% positive cells (2 points); 51%–75% positive cells (3 points); 76%–100% positive cells (4 points). Stained samples were analyzed under microscope (Aperio XT, LEICA, Germany). The total immunoreactive score was evaluated independently by two pathologists by summing by the nuclear and cytoplasm/membrane staining scores. For cases where the scores of two pathologists are inconsistent, a re-evaluation score should be made to reach a consensus.
5
Histochemical Analysis of Gal1GalNAc1 in CCA Tissues
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Biotinylated peanut agglutinin (PNA) (Vector Laboratories, Burlingame, CA, USA)
was used for detection of Gal1GalNAc1. Paraffin sections of CCA tissues were
deparaffinized in xylene, and then hydrated in downgraded ethanol and distilled
water. The antigens were unmasked by heating each section (in 0.1 mol/L citrate
buffer, pH of 6.0) in a pressure cooker. The sections were then treated with
absolute methanol containing 5% hydrogen peroxide for 30 minutes at room
temperature. After washing with phosphate-buffered saline and blocking
nonspecific binding (20% normal horse serum for 30 minutes at room temperature),
sections were (a) incubated with biotinylated lectin PNA (dilution of 1:20) for
2 hours at room temperature, and then (b) incubated with the VECTASTAIN® ABC HRP
Kit (Vector Laboratories) according to the manufacturer’s instructions. The
sections were visualized with 3,3ʹ-diaminobezidine-tetrahydrochloride, liquid
DAB+ (Dako, Carpinteria, CA, USA), and counterstained with hematoxylin and
eosin. The staining results were evaluated as the frequency of PNA-positive
cells at the tumor area. Staining was classified into four scoring categories as
follows: 0, negative; 1+, 1% to 10%; 2+, 11% to 50%; and 3+, >50%. Scores of
0 and 1+ were categorized as low expression and scores of 2+ and 3+ as high
expression for statistical analysis.
was used for detection of Gal1GalNAc1. Paraffin sections of CCA tissues were
deparaffinized in xylene, and then hydrated in downgraded ethanol and distilled
water. The antigens were unmasked by heating each section (in 0.1 mol/L citrate
buffer, pH of 6.0) in a pressure cooker. The sections were then treated with
absolute methanol containing 5% hydrogen peroxide for 30 minutes at room
temperature. After washing with phosphate-buffered saline and blocking
nonspecific binding (20% normal horse serum for 30 minutes at room temperature),
sections were (a) incubated with biotinylated lectin PNA (dilution of 1:20) for
2 hours at room temperature, and then (b) incubated with the VECTASTAIN® ABC HRP
Kit (Vector Laboratories) according to the manufacturer’s instructions. The
sections were visualized with 3,3ʹ-diaminobezidine-tetrahydrochloride, liquid
DAB+ (Dako, Carpinteria, CA, USA), and counterstained with hematoxylin and
eosin. The staining results were evaluated as the frequency of PNA-positive
cells at the tumor area. Staining was classified into four scoring categories as
follows: 0, negative; 1+, 1% to 10%; 2+, 11% to 50%; and 3+, >50%. Scores of
0 and 1+ were categorized as low expression and scores of 2+ and 3+ as high
expression for statistical analysis.
6
Immunofluorescence Staining of Cells
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Cells were grown on cover-slips. Wash cells twice with PBS and fix in 3.5% paraformaldehyde. Wash cells with PBS for 3 times. Cells are permeablized in PBS supplemented with 10% goat serum and 0.3% Triton X-100 for 15mins. Wash cells twice by PBS. Then cells were treated with 3% H2O2 for 10mins and wash for twice. Block cells with blocking buffer(10% goat serum in 1xPBS) for 1hr. Incubate cells with primary antibody(1:50) in blocking buffer for 1.5hr. Wash cells for 3 times by PBS. For γH2AX staining, the cells were incubated with Alexa Fluor® 488 Goat Anti-Mouse IgG for 1hr. After washing for 3 times, mount cover slips using VECTASHIELD with DAPI. For CtBP1 and CtBP2 staining, the Cells were incubated with EnVision+ System- HRP Labelled Polymer Anti-mouse (Dako, Carpinteria, CA, K4000) for 1 hr. After washing for 3 times, cells were incubated with Liquid DAB+ (Dako) for 3 minutes, and wash twice again. Then the cells were counterstained in Hematoxylin (Vector) for 30 sec, twice. Sequentially wash cells with water(twice, 5mins), 95% ethanol(2min) and 100% ethanol(2min). Finally, dip cells in xylene and mount with Permount onto a slide for microscopy imaging.
7
Immunohistochemical Analysis of Lung Tissue
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Lungs were fixed in 4% formalin for 24 h. After fixation, samples were dehydrated and embedded in paraffin. Sections 4-µm thick were stained with hematoxylin and eosin for microscopy examination and consecutive sections were used for immunohistochemical labeling. Sections were incubated with anti-ADAMTS12 (H-142, Santa Cruz Biotechnologies, 1 h at 37 °C; 1:50 dilution) or with anti-Ki-67 (ab66155, Abcam, o/n at 4 °C; 1:4000 dilution) primary antibodies. Sections were then incubated 30 min with EnVision™+/HRP (Dako) and 5 min with Liquid DAB (Dako). Samples were counterstained with hematoxilin.
8
Ex Vivo Amyloid-Beta Binding Assay
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To determine the ex vivo Aβ-binding ability of the NP-Ab, slides with APP/PS1 and WT mouse brain sections were deparaffinized and rehydrated for immunohistochemical staining using the EnVision G/2 Doublestain System, Rabbit/Mouse (DAB+/Permanent Red, K5361, Agilent Dako, Santa Clara, CA, USA), as per the manufacturers’ instructions. Adjacent sections on the same slide were incubated with either NP-Ab, rabbit polyclonal anti-Aβ antibody (1:600) as a positive control, or 1x PBS as a negative control for 20 min, followed 3,3′diaminobenzidine (liquid DAB+, DAKO, Glostrup, Denmark) for 5 min. Following three washes in 1x PBS, the slides were mounted with mounting medium and visualized using fluorescence microscopy (Zeiss AXIO microscope, Oberkochen, Germany).
9
Immunohistochemical HER2 Staining Protocol
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For the immunohistochemical HER2 staining, FFPE tissue sections were prepared and stained using the Dako Autostainer Universal Staining System (Dako). 2.5 μm thick sections of the FFPE samples were prepared and mounted onto silated microscopy slides (HistoSil slides, Stölzle-Oberglas). The dried slides were deparaffinated and rehydrated by immersion in xylene followed by immersion in ethanol of decreasing concentration (96 %, 80 %, 70 %). Epitope demasking was performed in a boiling citrate buffer pH 6 (Dako REALTM Target Retrieval Solution, Dako). Staining for HER2 was performed using a rabbit polyclonal antibody directed against ERBB2/HER2 (Dako, A0485) diluted 1:500 in the EnVisionTM FLEX Antibody Diluent (Dako). As a secondary antibody a horseradish peroxidase coupled polymer (EnVision+, Dako) was applied, which reacts with the substrate chromogen 3,3′-diaminobenzidinetetrahydrochloride (Liquid DAB+, Dako). Counterstaining for nuclei was performed using haematoxylin (Dako).
10
Immunohistochemical Analysis of uPAR
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Renal biopsy sections were fixed in paraformaldehyde for immunohistochemistry, and peroxidase and protein blockage was done using Novolink blocker for 50 minutes each. Then, human anti-uPAR antibody (1 : 50) was incubated overnight at 4°C. After, slides were incubated with Post Primary (Novolink Polymer Detection System Kit, BL, UK) for 50 minutes at room temperature and then incubated with the polymer (Novolink Polymer Detection System Kit, BL, UK) for 50 minutes. The material was then allowed to react with DAB substrate for staining (1,4-dideoxy-1,4-imino-D-arabinitol-diaminobenzidine) (Liquid DAB, Dako, Carpinteria, CA, USA) for 2 minutes, and sections were counterstained with hematoxylin and analyzed using a light microscope.
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