Retinal sections containing the optic nerve were fixed using 2% paraformaldehyde and incubated overnight in Brn3A antibody (1:25 dilution, Santa Cruz, CA) at 4 °C. Sections were blocked for 1 h with 20% goat serum in PBS containing 0.3%Triton X-100 and incubated for 2 h with secondary antibody (1:500) in PBS-containing 0.3% Triton X-100 and 1% BSA. Sections were cover-slipped with Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) and images were captured by fluorescent microscope (AxioObserver.Z1; Zeiss, Jena, Germany). The total number cells in the ganglion cell layer (GCL) were counted as DAPI-positive and retinal ganglion cells were counted as both Brn3A and DAPI-positive from one periphery of one Ora-Serrata to the other as described before [19 (link)].
Axioobserver z1
Manufactured by Vector Laboratories
Sourced in United States
The AxioObserver.Z1 is a high-performance inverted fluorescence microscope designed for advanced imaging and analysis. It features a motorized stand with automated components for precise control and reproducibility. The system is capable of a wide range of imaging techniques, including brightfield, phase contrast, DIC, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Brn3a antibody, by Santa Cruz Biotechnology (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Apoptag fluorescein in situ apoptosis detection kit, by Merck Group (1 mentions)
Vectashield without dapi, by Vector Laboratories (1 mentions)
Ax70 microscope, by Olympus (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Anti iba1 polyclonal antibody, by Fujifilm (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Alexa fluor 594, by Abcam (1 mentions)
Normal goat serum ngs, by Vector Laboratories (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
H 1000, by Vector Laboratories (1 mentions)
5 protocols using axioobserver z1
1
Fluorescent Quantification of Retinal Ganglion Cells
2
Apoptosis Detection in Retinal Sections
3
Immunohistochemical Analysis of Rat Sensorimotor Cortex
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We performed immunohistochemistry at P 17 (n = 4) and P 28 (n = 4). Under deep anesthesia with pentobarbital (>50 mg/kg), rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). The brains were obtained, post-fixed with the same fixative overnight, and cryoprotected with 30% sucrose. Coronal-sections (40 μm) were prepared from 1–3 mm posterior to the bregma that contains the sensorimotor cortex of the hindlimb area. After soaking with 0.25% Triton X-100 in PBS (PBS-T), blocking was performed with 10% normal goat serum (NGS) (Vector Labs, USA) for 60 min. The slices were reacted with anti-Iba1 polyclonal antibody (1:1,000; Wako, Japan) immersed in PBS-T containing 1% NGS at 4°C overnight, followed by immersion goat anti-rabbit IgG conjugated with Alexa Fluor 594 (1:1,000; Abcam, UK). After the slices were embedded on a glass slide with mounting medium (Vector Labs, USA), slices were photographed with a fluorescence microscope (Axio Observer.Z1; Zeiss, Germany) and an AX70 microscope (Olympus, Japan).
4
Transfection and Immunofluorescence of DmHsp27
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Hela cells were maintained in MEM Alpha (Gibco) supplemented with 5% FBS. Cells were plated in advance at a confluence of 175 000 cells/well (6 well plate) containing glass coverslip for transfection. Later, cells were incubated for 4 h in OptiMEM (Gibco) containing the plasmid pcDNA3.1(+)-DmHsp27:Lipofectamine (Invitrogen) complex (1.5 μl Lipofectamine/1.5 μg DNA), cells were washed with culture medium and incubated for 48 h to express DmHsp27 before immunofluorescence.
Immunofluorescence was performed as described in [35 (link), 78 (link)]. Briefly, cells were washed with PBS and fixed in methanol at − 20 C for 20 min. Cells were blocked in PBS 0.1% Tween20-X (PBST) containing 5% BSA (PBST-BSA) and were incubated one hour at room temperature with primary antibody (monoclonal anti-DmHsp27 (2C8E11) [34 (link), 35 (link)]) diluted (1/20) in PBST-BSA. After they, were washed with PBST and were incubated 45 min with secondary antibody (goat anti-mouse Alexa 488 (Invitrogen)). Finally, cells were mounted in Vectashield mounting medium (Vector Laboratories) and examined using fluorescence microscopy (Axio Observer Z1).
Immunofluorescence was performed as described in [35 (link), 78 (link)]. Briefly, cells were washed with PBS and fixed in methanol at − 20 C for 20 min. Cells were blocked in PBS 0.1% Tween20-X (PBST) containing 5% BSA (PBST-BSA) and were incubated one hour at room temperature with primary antibody (monoclonal anti-DmHsp27 (2C8E11) [34 (link), 35 (link)]) diluted (1/20) in PBST-BSA. After they, were washed with PBST and were incubated 45 min with secondary antibody (goat anti-mouse Alexa 488 (Invitrogen)). Finally, cells were mounted in Vectashield mounting medium (Vector Laboratories) and examined using fluorescence microscopy (Axio Observer Z1).
5
Immunohistochemical Analysis of Tumor-Bearing Mice
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Tumor-bearing mice were perfused with PBS and subsequently with 4% paraformaldehyde. Brains and spines were harvested, followed by sectioning for histological analyses. Brain and spine sections on slides were washed in PBS and mounted with aqueous mounting medium (#H1000 and #H1200, Vector Laboratories) to be visualized with confocal microscopy (Axio Observer.Z1, Zeiss). For fluorescence immunohistochemistry, sections were incubated with primary antibodies overnight at 4°C. After wash, secondary antibodies were probed and detected by confocal microscope. For hematoxylin and eosin (H&E) staining, sections were incubated with H&E Y dye (1% alcohol), dehydrated with 70, 95, and 100% ethanol, and mounted in xylene-based mounting medium.
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