Mice were anaesthetised with isoflurane and transcardially perfused with saline, followed by 4 % paraformaldehyde (PFA) and had their brains extracted. Brains were fixed overnight in PFA at 4 °C. After fixation, brains cryo-protected in a 30 % sucrose solution at 4 °C for 1–2 days. Brains were then frozen in FSC Clear (Leica) and sliced using a cryotome (Leica, CM1850) at 30 μm thickness. Slices were incubated in a blocking solution (2 % goat serum, 0.3 % Triton-X, in 0.1 M PBS) for 1 hour at room temperature on a shaking plate. Slices were then incubated in primary antibodies, 1:1,000 rabbit anti-CGRP (Bachem, T4032) or 1:1,000 rabbit anti-serotonin (Immunostar, 20080) solution for 17 hours at 4 °C. Slices were then washed for 10 minutes, three times in PBS. Slices were then incubated in a secondary antibody solution for 1 hour. Secondary antibody, Goat anti-rabbit 647 (Molecular Probes, A21245) was used at 1:200 dilution. Slices were then washed for 10 minutes, three times in PBS. DAPI was added (1:10,000) during the second PBS washing. Slices were then mounted on a microscope glass and mounted with DAKO mounting media. Z stack images were acquired with a confocal microscope (Zeiss, LSM780) and maximum intensity Z projected through ImageJ (NIH) software.
Fsc clear
Manufactured by Leica
The FSC Clear is a technical laboratory equipment product offered by Leica. It serves as a clearing agent, facilitating the preparation and processing of biological samples for microscopic analysis. The core function of the FSC Clear is to improve the transparency and clarity of specimen samples, enabling enhanced visualization and examination.
Automatically generated - may contain errors
2 protocols using fsc clear
1
Immunofluorescent Labeling of CGRP and Serotonin in Mouse Brain
2
Immunofluorescent Labeling of CGRP and Serotonin in Mouse Brain
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Mice were anaesthetised with isoflurane and transcardially perfused with saline, followed by 4 % paraformaldehyde (PFA) and had their brains extracted. Brains were fixed overnight in PFA at 4 °C. After fixation, brains cryo-protected in a 30 % sucrose solution at 4 °C for 1–2 days. Brains were then frozen in FSC Clear (Leica) and sliced using a cryotome (Leica, CM1850) at 30 μm thickness. Slices were incubated in a blocking solution (2 % goat serum, 0.3 % Triton-X, in 0.1 M PBS) for 1 hour at room temperature on a shaking plate. Slices were then incubated in primary antibodies, 1:1,000 rabbit anti-CGRP (Bachem, T4032) or 1:1,000 rabbit anti-serotonin (Immunostar, 20080) solution for 17 hours at 4 °C. Slices were then washed for 10 minutes, three times in PBS. Slices were then incubated in a secondary antibody solution for 1 hour. Secondary antibody, Goat anti-rabbit 647 (Molecular Probes, A21245) was used at 1:200 dilution. Slices were then washed for 10 minutes, three times in PBS. DAPI was added (1:10,000) during the second PBS washing. Slices were then mounted on a microscope glass and mounted with DAKO mounting media. Z stack images were acquired with a confocal microscope (Zeiss, LSM780) and maximum intensity Z projected through ImageJ (NIH) software.
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