Cohorts of mice were sacrificed at age 12 weeks ± 3 days. Age-matched cohorts that included males and females of the two genotypes of interest (WT and S1782E+/−) were processed together throughout. Vaginal cytology was collected on all female mice at the time of sacrifice. Mice were sacrificed by lethal CO2 inhalation followed by decapitation and whole brain extraction. Golgi staining was performed using the FD Rapid GolgiStain Kit (FD Neurotechnologies, Inc) according to manufacturer’s instructions. Brains were sectioned (150 µm) on a cryostat. Tissue sections were placed onto gelatin-coated slides and allowed to dry overnight at room temperature. The following day, sections were then rehydrated with distilled and deionized water, reacted in a developing solution (FD Rapid GolgiStain Kit, FD Neurotechnologies), and dehydrated through graded ethanols. Finally, sections were cleared in xylene and coverslipped using Permount (Fisher Scientific). Auditory cortex was defined using gross anatomical landmarks and alignment with the corresponding Nissl plate containing A1 in the mouse brain atlas [96 ]. Within A1, deep layer 3 was defined as 30–50% of the distance between the pial surface and white matter border and sampling of pyramidal cells was performed as described below.
Fd rapid golgistain kit
Manufactured by FD NeuroTechnologies
Sourced in United States
The FD Rapid GolgiStain Kit is a laboratory tool designed for the rapid and efficient staining of neuronal morphology in biological samples. It utilizes a modified Golgi staining technique to selectively visualize the intricate structures of individual neurons within a given tissue.
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Lab products found in correlation
Vt1000, by Leica (22 mentions)
Permount, by Thermo Fisher Scientific (21 mentions)
Neurolucida software, by MicroBrightField (12 mentions)
Vibratome, by Leica (11 mentions)
Neurolucida, by MBF Biosciences (9 mentions)
Eclipse ci l microscope, by Nikon (9 mentions)
Cryostat, by Leica (9 mentions)
Vt1200, by Leica (9 mentions)
Axio observer z1, by Zeiss (9 mentions)
Gelatin coated slides, by FD NeuroTechnologies (8 mentions)
Neurolucida software, by MBF Biosciences (8 mentions)
Neurolucida 360, by MBF Biosciences (8 mentions)
Vt1200s vibratome, by Leica (7 mentions)
Image pro plus 6, by Media Cybernetics (7 mentions)
Vibratome vt1000, by Leica (5 mentions)
Cm1950, by Leica (5 mentions)
Neurolucida explorer, by MBF Biosciences (4 mentions)
Bx61 microscope, by Olympus (4 mentions)
Fv1000, by Olympus (4 mentions)
Neuroexplorer, by MBF Biosciences (4 mentions)
503 protocols using fd rapid golgistain kit
1
Golgi Staining of Auditory Cortex
2
Golgi Staining of Mouse Auditory Cortex
3
Golgi Staining Protocol for Spine Analysis
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Golgi staining was adopted to determine the changes of spines according to the manufacturer's instructions (FD Rapid GolgiStain TM kit, FD NeuroTechnologies, Inc). Briefly, the fresh rat brains were incubated with pre-prepared impregnation solution at room temperature for 2 weeks in the dark. Thereafter, the collected brains were then incubated with manufacturer-provided solution C for another 96 h at room temperature. The brain sections of 200 μm were then prepared and mounted with solution C on gelatin-coated slides and allowed to dry naturally for 2 days. Thereafter, the brain sections were then rinsed and incubated with a mixture of manufacturer-provided solutions D and E for 10 min. The brain sections were subsequently rinsed, dehydrated, and cleared with xylene and ethanol. The images were captured using an OLYMPUS IX70 microscope. The ImageJ software was used to process and analyze the images.
4
Golgi Staining of Cortical Pyramidal Neurons
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Golgi staining was performed using FD Rapid GolgiStainTM kit according to its manufacturer’s protocol (FD NeuroTechnologies, PK401, Columbia, MD). Briefly, brains were immersed in impregnation solution (a mixture of FD Solution A:B = 1:1) for 2 weeks at room temperature in the dark. Then brains were immersed in FD Solution C and preserved in the dark at room temperature for 48–72 h. The brain tissues were coronally sliced (150 μm) using Leica CM1950 cryostat and mounted on 0.5% gelatin-coated slides. The sections were then stained with the staining solution according to manufacturer’s protocol and coverslipped using Permount medium. Images of cortical pyramidal neurons were captured using Olympus BX51 with Cell sens dimension 1.12 software (Olympus) at random.
For pyramidal neuron spine density analysis, the number of spines was counted on the following segments: for apical dendrites, dendritic segments (20 μm) located further than 100 μm from cell soma of pyramidal neurons were quantified.
For pyramidal neuron spine density analysis, the number of spines was counted on the following segments: for apical dendrites, dendritic segments (20 μm) located further than 100 μm from cell soma of pyramidal neurons were quantified.
5
Quantifying Dendritic Spines in Mice
6
Golgi-Cox Staining for Dendritic Spine Analysis
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For Golgi-Cox staining, 6-month-old mice were sacrificed by rapid decapitation and their brains were removed. The brains were immersed in FD Rapid Golgi StainTM kit solution (FD Neurotechnologies, PK401) and processed according to the manufacturer’s instructions. Then, the tissues were sliced to 60 μm and stained according to the manufacturer’s protocol. For quantification of dendritic spines, primary auditory cortexes were imaged using a CSU-X spinning disc confocal microscope (Intelligent Imaging Innovations) with an HQ2 CCD camera (Photometrics) using a 100x objective. Four or 5 dendritic segments per cell were analyzed and the spine density is presented as the mean number of spines per 10 µm of dendritic length, calculated using a double-blind method. Experiments were independently repeated with 3 mouse brains each group.
7
Quantifying Synaptic Structural Plasticity in Spinal Cord Neurons
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To observe synaptic structural plasticity, we used Golgi-Cox staining and
counting of dendritic spines as previously described.37 (link) Staining was performed
according to the manufacturer’s instructions of the FD Rapid Golgi Stain
TM Kit (FD Neurotechnologies, Inc., Columbia, USA). Briefly, the
spinal cord tissue was immersed in solutions A and B for 24 h, then the solution
was changed and the samples stored in the dark at room temperature for 2 weeks.
The spinal cord tissue was then stored in solution C in the dark at 4°C for
another 72 h. A cryostat was used to prepare spinal cord slices and an optical
microscope to image and analyze dendrites and dendritic spines in the dorsal
horn of the spinal cord. Assessment of the overall complexity of WDR neurons by
Sholl analysis. First, we used the Neuron ImageJ software to track the WDR
neurons, got a 2D distribution map of neurons, then drew a concentric circle
every 20 μm with the neuron cell body as the center, recorded the number of
intersection points between each concentric circle and dendrites. We also
measured the perimeter of the cell body, the total length of dendrites, the
number of primary branches, and the percentage of primary branches with
secondary branches to analyze the changes in neuron cell body and dendritic
branches.
counting of dendritic spines as previously described.37 (link) Staining was performed
according to the manufacturer’s instructions of the FD Rapid Golgi Stain
TM Kit (FD Neurotechnologies, Inc., Columbia, USA). Briefly, the
spinal cord tissue was immersed in solutions A and B for 24 h, then the solution
was changed and the samples stored in the dark at room temperature for 2 weeks.
The spinal cord tissue was then stored in solution C in the dark at 4°C for
another 72 h. A cryostat was used to prepare spinal cord slices and an optical
microscope to image and analyze dendrites and dendritic spines in the dorsal
horn of the spinal cord. Assessment of the overall complexity of WDR neurons by
Sholl analysis. First, we used the Neuron ImageJ software to track the WDR
neurons, got a 2D distribution map of neurons, then drew a concentric circle
every 20 μm with the neuron cell body as the center, recorded the number of
intersection points between each concentric circle and dendrites. We also
measured the perimeter of the cell body, the total length of dendrites, the
number of primary branches, and the percentage of primary branches with
secondary branches to analyze the changes in neuron cell body and dendritic
branches.
8
Golgi Staining Technique for Mouse Brain
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The FD Rapid GolgiStainTM Kit (PK401, FDNeuroTechnologies, INC., Columbia, USA) was used for Golgi staining. Three Ctrl and three cKO mice were sacrificed at P14, P17 and P21, and the brain tissues were quickly removed. The tissues were immersed in the mixed solution (A:B = 1:1) and then placed in a dark place at room temperature for 3 weeks. Then, the tissues were transferred into solution C in a dark room at room temperature for 3 days. The tissues were cut into 100 µm sections, and each section was dried at room temperature. The sections were washed in double-distilled water (ddH2O) twice for 3–4 min each and then placed in a mixture (D:E:ddH2O = 1:1:2) for 10 min. The sections were immersed in 50, 75, 95 and 100% ethanol for 4 min each. Lastly, the sections were cleared in xylene three times for 4 min each and, finally, sealed with neutral resin.
9
Dendritic Spine Analysis in Aging Mice
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Control (n = 5) vs. TgCPEB4Δ4 mice (n = 4) three month-old mice, and WT (n = 3) vs. CPEB4 KOGT/+ (n = 4) fifteen month-old were completely anesthetized with an intraperitoneal pentobarbital injection (60 mg/kg Dolethal®, Vetoquinol). The whole brain was extracted and immersed in Golgi-Cox staining solution (FD Rapid GolgiStainTM kit, FD Neurotechnologies, cat. PK401). 150 µm sagittal sections were obtained in a Leica VT1200S vibratome and mounted on gelatin-coated slides. Golgi staining was performed as manufacturer’s instructions. Afterwards, all sections were counterstained with Toluidine Blue pH 4.0 (1 g/l Toluidine Blue (Sigma, 198161), 0.8 M glacial acetic acid) and coverslipped with DePeX (Amsbio, 18243.02). Pyramidal neurons from layer II/III of the cortex were identified by their distance from pia mater and their distinct morphologies. Secondary, tertiary and quaternary dendrites of these neurons were selected for analysis. Z-stacks of the entire apical dendritic tree of Golgi stained pyramidal neurons (up to 80 μm total on Z-axis, optical section thickness = 0.5 μm) were taken at 40x magnification with 2x optical zoom on a vertical Zeiss Axio Imager.Z1 M and analyzed by Laser Scanning Microscope LSM 510 v.4.2 SP1 (Carl Zeiss). Spine density, length and classification were performed according to58 (link), unbiased blinded to genotype.
10
Rapid Golgi Staining for Detailed Neuroanatomy
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Animals were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) before euthanizing. Brains were removed quickly from the skull to avoid any damage. After rinsing, tissue was sliced in ~10 mm thick blocks. The blocks were stained with the FD Rapid Golgi StainTM kit (FD Neuro Technologies, USA). Briefly, the blocks were first immersed in the impregnation solution (A and B), which was replaced after 12 h and then kept in dark for 14 days. Afterward, the blocks were put in solution C, which was replaced after 24 h and kept in dark for the next 48–60 h. Then the blocks were sliced on a cryomicrotome at 100 μm. Slices were mounted on agelatin-coated microscope slides, stained, and dehydrated and cover slipped with Permount.
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