For potential unmasking of Pgp epitopes, the sections were pretreated with a mixture of 33% acetic acid and 66% ethanol for 10 minutes. Then the sections were washed in trisbuffered saline (TBS) and blocked for 60-min with 3% bovine serum albumin (BSA) and 11% normal rabbit serum (NRS) in TBS as a blocking solution. The primary antibody (monoclonal mouse anti-Pgp, C219, Dako) was diluted (15 μg/ml) in 1% BSA and 1% NRS. Sections were incubated overnight at 4°C. After washing in TBS, the sections were incubated with biotinylated rabbit anti-mouse antibody (Dako) in 1:200 for 60 min, followed by 90-min incubation with horseradish peroxidase–labeled streptavidin (1.65 μg/ml) at room temperature. 3, 3-Diamino-benzidine (DAB Peroxidase Substrate Kit, Vector Laboratories) was used for visualization. Histological analysis was performed on at least three stained sections of every brain.
Biotinylated rabbit anti mouse antibody
Manufactured by Agilent Technologies
Biotinylated rabbit anti-mouse antibody is a laboratory reagent used in various bioassays and immunodetection techniques. It is a secondary antibody that binds to mouse primary antibodies, with biotin conjugated to facilitate further detection or signal amplification.
Automatically generated - may contain errors
Lab products found in correlation
Normal swine serum, by Agilent Technologies (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Eukitt, by Merck Group (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Anti cd68 antibody, by Agilent Technologies (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Prolong gold anti fade reagent with 4, by Thermo Fisher Scientific (1 mentions)
Liquid permanent red, by Agilent Technologies (1 mentions)
Dual endogenous enzyme blocking reagent, by Agilent Technologies (1 mentions)
Liquid permanent red substrate chromogen, by Agilent Technologies (1 mentions)
5 protocols using biotinylated rabbit anti mouse antibody
1
Pgp Epitope Unmasking Protocol
2
Immunohistochemical Analysis of Mouse Tissues
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After sacrifice, mouse organs were fixed in 4% buffered formaldehyde for 24 h, rinsed with PBS, dehydrated in a series of graded ethanol and embedded in paraffin. Sections of 5 μm thickness were cut and stained with haematoxylin and eosin. For immunohistochemistry 5 μm thick sections were cut, dewaxed, microwaved in Target Retrieval Solution (DAKO) for 2 x 4 min and cooled down to room temperature for 40 min. After washing with Tris-buffered saline (TBS), non-specific binding was blocked by incubating sections in 10% normal swine serum (DAKO) for 30 min at room temperature. Slides were incubated with anti-CD68 antibody (ABCAM ab955) at a dilution of 1 μg/ml for 60 min (RT), followed by a biotinylated rabbit anti mouse antibody (DakoCytomation) at a dilution of 1:200 for 30 min. After careful washes in TBS, an incubation with an avidin-alkaline phosphatase complex (ABC kit, Vectastain, Vector) for 30 min followed and thereafter, additional washes in TBS were performed. Alkaline phosphatase activity was visualized using Liquid Permanent Red (LPR) Substrate-Chromogen (DAKO) for 15 min. After washing with water, slides were counterstained with Mayer's hemalum diluted 1:1 in water for ten seconds, blued under water and mounted with Eukitt® (Sigma).
3
Antibody-based Protein Detection Methods
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Antibodies were used for Western immunoblots, indirect immunofluorescence microscopy, and immunohistochemistry as per manufacturer's instructions: paxillin mouse antibody (P13520; Transduction Laboratories, San Jose, CA), FLI-1 rabbit antibody (15289; Abcam, Cambridge, MA), zyxin rabbit antibody (B71; Beckerle Lab), α5 integrin (610633; BD Bioscience, San Jose, CA), β-actin AC-74 mouse antibody and vinculin V9131 mouse antibody (Sigma-Aldrich, St. Louis, MO), α-tubulin mouse antibody (12G10; Developmental Studies Hybridoma Bank, Iowa City, IA), and CD99 (clone 0-13 mouse antibody; 620-01; Signet, Cambridge, UK). Secondary antibodies were horseradish peroxidase (HRP)–conjugated antibodies for immunoblots (GE Healthcare, Pittsburgh, PA), the Alexa Fluor antibodies and phalloidin for microscopy (Molecular Probes/Invitrogen, Grand Island, NY), and biotinylated rabbit anti-mouse antibody (E0413; Dako, Carpinteria, CA) for immunohistochemistry. Fibronectin to coat coverslips and Mowiol to mount coverslips (Sigma-Aldrich) and Vybrant Cell-Labeling solutions DiI and DiO (Molecular Probes/Invitrogen) were used as per manufacturer's instructions. d -Luciferin (Luck; Gold Biotech, St. Louis, MO) was injected for bioluminescence detection. Prolong Gold anti-fade reagent with 4′,6-diamidino-2-phenylindole (DAPI; P-36931; Invitrogen/Life Technologies) was used for mounting lung sections.
4
Dual Immunohistochemical Staining of p53 and GLuc
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Formalin-fixed tumour tissue was embedded in paraffin, cut and fixed on glass
slides overnight at 37 °C. Upon antigen retrieval with
Tris-EDTA pH 9.0, sections for double staining were blocked in Dual Endogenous
Enzyme Blocking Reagent (Dako) and incubated with α-p53 antibody (DO-1, 1:1,000) in
Antibody Diluent (Dako REAL) overnight at 4 °C. Biotinylated
rabbit-anti-mouse antibody (Dako, E0464, 1:500) served as secondary antibody and
was incubated with Streptavidin-labelled Peroxidase (KPL) followed by detection
with DAB Plus Reagent Set (Life Technologies). For subsequent GLuc staining, sections were incubated
with α-GLuc antibody (Nanolight Technologies 401 P, 1:1,000) at
4 °C overnight. Biotinylated goat-anti-rabbit antibody
(Dako, E0432, 1:500) was used as secondary antibody and was detected with
Phosphatase-labelled Streptavidin (KPL) and Liquid Permanent Red (Dako). Nuclei
were counterstained with Mayersches Haemalaun (Merck) for 15 s before
fixation in Mowiol.
slides overnight at 37 °C. Upon antigen retrieval with
Tris-EDTA pH 9.0, sections for double staining were blocked in Dual Endogenous
Enzyme Blocking Reagent (Dako) and incubated with α-p53 antibody (DO-1, 1:1,000) in
Antibody Diluent (Dako REAL) overnight at 4 °C. Biotinylated
rabbit-anti-mouse antibody (Dako, E0464, 1:500) served as secondary antibody and
was incubated with Streptavidin-labelled Peroxidase (KPL) followed by detection
with DAB Plus Reagent Set (Life Technologies). For subsequent GLuc staining, sections were incubated
with α-GLuc antibody (Nanolight Technologies 401 P, 1:1,000) at
4 °C overnight. Biotinylated goat-anti-rabbit antibody
(Dako, E0432, 1:500) was used as secondary antibody and was detected with
Phosphatase-labelled Streptavidin (KPL) and Liquid Permanent Red (Dako). Nuclei
were counterstained with Mayersches Haemalaun (Merck) for 15 s before
fixation in Mowiol.
5
Immunohistochemical Analysis of Lrp1 in Tissue
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Knees were fixed in 3.7% formalin as described above and embedded in paraffin. Five-μm-thick sections were cut, dewaxed, microwaved in Target Retrieval Solution (DAKO) for 2 × 4 min and cooled down to RT for 40 min. After washing with TBS, slides were blocked by incubating sections in 10% normal swine serum (DAKO) for 30 min at RT. Then, the slides were incubated with anti-Lrp1 antibody (polyclonal LRP1-377, provided by J.Her.) or rabbit control antibody at a dilution of 1 µg·mL-1 for 60 min at RT, followed by a biotinylated rabbit anti-mouse antibody (DakoCytomation) at a dilution of 1:200 for 30 min. Hereafter, the slides were washed with TBS, incubated with an avidin–alkaline phosphatase complex (ABC kit, Vectastain, Vector) for 30 min, and washed 3× with TBS. Alkaline phosphatase activity was visualized using Liquid Permanent Red Substrate-Chromogen (DAKO) for 15 min. After washing with water, slides were counterstained with Mayer’s hemalum diluted 1:1 in water for 10 s, blued under water, and mounted with Eukitt® (Sigma).
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