DAB-peroxidase IHC was performed as described by Kwakowsky et al. (2018 (link)). In brief, sections were washed in PBS with 0.2% Triton X-100 (PBST) before blocking for endogenous peroxidases (50% methanol and 1% H2O2) for 20 min, followed by three 10-min washes in PBST and incubated for 72 h in primary antibody in immunobuffer at 4°C (Table 3 ). Following three 10-min washes in PBST the sections were incubated for 24 h with the biotinylated secondary antibody (anti-mouse IgG-Biotin antibody produced in goat 1:1,000) in immunobuffer at room temperature (RT). The sections were then washed in PBST before incubation with ExtrAvidin (1:1,000, E2886; Sigma, St. Louis, MO, USA) in immunobuffer for 4 h at RT, followed by three 10-min washes in PBST before development in 0.05% DAB and 0.01% H2O2 in 0.1 M phosphate buffer. Sections were washed in PBST, mounted onto glass slides, dried, dehydrated through a graded series of ethanol, and cleared in xylene. The slides were coverslipped with DPX mountant (1019790500; Merck, Whitehouse Station, NJ, USA). The sections were imaged on either a Leica DMRB light microscope or a Leica MZ6 dissecting microscope (Wetzlar, Germany).
Mz6 dissecting microscope
Manufactured by Leica
Sourced in Germany
The Leica MZ6 is a dissecting microscope designed for visual observation and analysis. It features a magnification range of 6.3x to 40x, allowing for detailed examination of specimens. The MZ6 includes a robust, ergonomic design and coaxial controls for smooth operation.
Automatically generated - may contain errors
Lab products found in correlation
Dpx mountant, by Merck Group (2 mentions)
Dmrb light microscope, by Leica (2 mentions)
Extravidin, by Merck Group (2 mentions)
Dfc450 camera, by Leica (1 mentions)
Lsm 800 airyscan, by Zeiss (1 mentions)
Apotome, by Zeiss (1 mentions)
Axiozoom, by Zeiss (1 mentions)
Ic90 e camera, by Leica (1 mentions)
4 protocols using mz6 dissecting microscope
1
Immunohistochemical Staining Protocol
2
Cranial Morphometry Analysis Protocol
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Gross anatomy was photographed with a Leica MZ6 dissecting microscope and images captured with a DFC450 camera before analysis using the open-source ImageJ software (v1.53). Histology slides were photographed on a Zeiss AxioScan seven and immunofluorescent sections on a Zeiss AxioZoom, Apotome or LSM800-Airyscan microscope equipped with Zen software (v2.3 or 3.0). Centroid size of crania was quantified in ImageJ by delimiting the shape above a virtual line from the upper jaw to earlobe to occiput in the sagittal plane (Pilatti and Astúa, 2017 (link)).
3
Immunohistochemistry Protocol for DAB-Peroxidase Staining
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DAB‐peroxidase IHC was performed as described by Kwakowsky et al. (27 (link)). In brief, sections were washed in PBS with 0.2% Triton X‐100 (PBST) before blocking for endogenous peroxidases (50% methanol and 1% H2O2) for 20 min, followed by three 10‐min washes in PBST and incubated for 72 h in primary antibodies in immunobuffer at 4℃ (Table 3 ). The sections were then washed in PBST before incubation for 24 h with the biotinylated secondary antibodies (anti‐Mouse IgG‐Biotin antibody produced in goat 1:1000, anti‐rabbit IgG‐Biotin antibody produced in goat 1:1000) in immunobuffer at room temperature (RT). The sections were then washed in PBST before incubation with ExtrAvidin (1:1000, E2886; Sigma, St. Louis, MO, USA) in immunobuffer for 4 h at RT, followed by three 10‐min washes in PBST before development in 0.05% DAB and 0.01% H2O2 and 0.1 M phosphate buffer. Sections were washed in PBST and mounted onto glass slides, dried, dehydrated through a graded series of ethanol, and cleared in xylene. The slides were coverslipped with DPX mountant (1019790500; Merck, Whitehouse Station, NJ, USA). The sections were then imaged on either a Leica DMRB light microscope or a Leica MZ6 dissecting microscope (Wetzlar, Germany).
4
Xenopus Oocyte Isolation and Characterization
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All procedures were performed at room temperature. Ovaries were removed from adult females (Xenopus One, MI, U.S.A.) according to a protocol approved by the Health Sciences Animal Care and Use Committee of the University of Alberta (AUP 00000942). In this protocol, animals are anesthetized using tricaine methanesulfonate and then euthanized by decapitation. Oocytes were released from their follicles by collagenase treatment (2 h with 3 mg/ml collagenase and 1 mg/ml bovine serum albumin [BSA] in OR‐2 medium; 5 mM Hepes‐NaOH pH 7.8, 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM Na2HPO4).35 Somatic cells still associated with oocytes were then removed by gentle rocking on a sandpaper surface under OR‐2 medium (Liu and Liu, 2006).36 DAPI staining (1 mg/ml) of randomly selected oocytes confirmed detachment of somatic cells. Oocytes were maintained in OR‐2 with penicillin and streptomycin (100 μg/ml each) until use within 3 day after isolation. They were imaged using a MZ6 dissecting microscope, IC90 E camera, and Application Suite v.4.12.0 (Leica). Oocyte diameter was estimated from these images.
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