Coronal sections of 25 μm in thickness were cut using cryostat (Leica CM3050 S, Leica Microsystems Nussloch, Nussloch, Germany) from the PFC of the brains (from 1.98 to 1.54 mm anterior to Bregma) for immunohistochemistry and immunofluorescent procedures. For detection of PV and NPY, standard immunohistochemistry staining procedures were performed (details provided in Supplementary Information ) with two different anti-PV (P3088 and SAB4200545; Sigma-Aldrich, St. Louis, MO, USA; both 1:500) as well as anti-NPY (ab10980; Abcam, Cambridge, UK; 1:500) as primary antibodies. For fluorescence imaging, tissues were visualized using an epifluorescent IX81 microscope (Olympus, Münster, Germany) and for confocal imaging a 700-AX10 laser scanning microscope (Carl Zeiss, Jena, Germany) was used.
700 ax10 laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss 700-AX10 is a laser scanning microscope designed for high-resolution imaging. It features a laser excitation source and a scanning system that allows for the capture of detailed images of samples. The core function of this microscope is to provide users with a tool for conducting advanced microscopic analysis and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Ab10980, by Abcam (1 mentions)
P3088, by Merck Group (1 mentions)
Cm3050, by Leica (1 mentions)
Ab31895, by Abcam (1 mentions)
Ab150077, by Thermo Fisher Scientific (1 mentions)
Ab13552, by Abcam (1 mentions)
Faramount aqueous mounting medium, by Agilent Technologies (1 mentions)
Ab150115, by Abcam (1 mentions)
3 protocols using 700 ax10 laser scanning microscope
1
Immunostaining of Prefrontal Cortex
2
Double-Immunofluorescence of TRPV1 and PSD95
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For double-immunofluorescence of TRPV1 and PSD95 (a marker for post synaptic mechanisms), free-floating sections containing PFC were washed in PBS and then preincubated in PBS containing 5% normal goat serum for 3–4 h at RT. After preincubation, sections were incubated with polyclonal rabbit anti-TRPV1 antibody (ab31895, Abcam, dilution: 1:500), and mouse monoclonal antibody anti-PSD95 (ab13552; Abcam; 1:500) for 24 h at 4°C, after which they were rinsed 3–5 times in PBS, the sections were then incubated for 1–2 h at RT in goat anti-rabbit IgG Alexa Fluor 488 (ab150077; Invitrogen; 1:1500) and goat anti mouse IgG Alexa Fluor 647 (ab150115; Abcam; 1:1500) dissolved in blocking solution. Following the final rinsing, the slices were wet mounted onto subbed slides and subsequently dried and cover-slipped with Dako Faramount Aqueous Mounting Medium (Dako; S3025). For fluorescence imaging, tissues were visualized under ab epifluorescence IX81 microscope (Olympus), and for confocal imaging a 700-AX10 laser scanning microscope (Carl Zeiss) was used.
3
Quantifying Cortical Cell Populations
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For quantification, the brain areas and the layer borders were defined according to the mouse brain atlas (Paxinos G, Franklin KBJ) and based on cytoarchitectural features as described before (Saffari et al., 2016 ) Multiple alignment of images taken with × 4 and × 10 magnifications was performed with CellˆP software (Olympus). Distributions of positively stained cells were analyzed using ImageJ software (NIH, Bethesda, MD, USA) in the anterior cingulated cortex (ACC), PrL and IL. For each region, mean numbers of cells as well as cell density (cell × mm−2) were calculated across all the layers in above regions.
To count and quantify GAD67 positive neurons and EPOR positive cells for immunofluorescent procedure, sections were viewed with confocal imaging 700-AX10 laser scanning microscope (Carl Zeiss). GAD67 positive neurons and EPOR positive cells were counted across all the layers in medial prefrontal cortex in order to calculate the percent co-localization of both markers. (more details about procedures s.Supplemental Information ).
To count and quantify GAD67 positive neurons and EPOR positive cells for immunofluorescent procedure, sections were viewed with confocal imaging 700-AX10 laser scanning microscope (Carl Zeiss). GAD67 positive neurons and EPOR positive cells were counted across all the layers in medial prefrontal cortex in order to calculate the percent co-localization of both markers. (more details about procedures s.
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