Brains from postnatal day 21 (P21) and P42 WT mice and KO mice were impregnated in Golgi solutions using the FD Rapid Golgi staining kit (FD Neurotechnologies, Columbia, MD) according to the manufacturer’s instructions. Coronal sections of 150 μm were made on a cryostat. After staining, images were captured with a ×40 objective on a Leica confocal laser scanning SP5 microscope with bright field settings. For spine density analysis, dendrite segments of 25–150 μm were randomly selected on the secondary apical or basal dendrite. According to the head diameter and spine length, dendritic spines were divided into three categories for quantification: mushroom type, thin type, and stubby type16 (link).
Fd rapid golgi staining kit
Manufactured by FD NeuroTechnologies
Sourced in United States
The FD Rapid Golgi staining kit is a laboratory tool designed to aid in the visualization of neuronal structures, particularly the dendritic morphology and dendritic spines. The kit provides a reliable and efficient method for impregnating neurons with silver salts, allowing for the detailed observation of their intricate features under a microscope.
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9 protocols using fd rapid golgi staining kit
1
Golgi Staining and Spine Analysis
2
Golgi-Cox Staining Reveals Dendritic Changes
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Golgi-Cox staining (FD Rapid Golgi Staining Kit; FD Neuro Technologies, United States) was performed to observe changes in dendrites in the infarcted penumbra cortex (Hu et al., 2020 (link)). Dendritic structures were analyzed by laser confocal scanning microscopy (Olympus FV1000, Japan). The Fiji software (https://imagej.net/Fiji ) was used for neuron tracking. Sholl analysis was conducted to analyze trajectory, automatically drawing a concentric circle with the cell body as the center, with a step of 10 μm. The complexity of dendrites was quantified by the number of intersections and the number of neuron branches.
3
Golgi-Cox Staining for Hippocampal Neuron Spines
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Brains were processed for Golgi-Cox staining using the FD Rapid Golgi Staining kit (FD Neurotechnologies, Inc). Brains were left in the staining solution for 12 days, frozen in isopentane solution and kept at −80 °C until sectioning and coloration. The brains were cryostat-cut at a thickness of 100 μm. Sections were mounted on gelatin-coated slides and analyzed using a motorized Leica DM5000 microscope at ×63 magnification. The images were acquired using a CCD Coolsnap camera and Metamorph software. For analysis, we randomly selected pyramidal neurons from the CA1 region of the dorsal hippocampus that were fully penetrated by the Golgi coloration and distinguishable from other neurons (n-3 deficient mice had less usable neurons than n-3 sufficient mice). Spine density in these neurons was determined by counting the number of spines on at least 3 basal and 3 apical dendritic segments of 10 μm in length. Segments from dendrites situated as far from the cell body as possible, with no overlap with other dendrites were randomly selected. Primary dendrites were never used for analysis as their thickness hampers detection of spines. Spine density was calculated per 10 μm and averaged across the different segments in the same neuron. Images were processed with Mercator Pro 7.9.11.
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Rapid Golgi Staining of Brain Tissue
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Golgi staining was performed according to the instructions of the FD rapid Golgi staining kit (FD NeuroTechnologies, America). In short, the prepared brain tissue was immersed in equal volume mixed solutions A and B, placed at room temperature away from light for two weeks, and replaced with a new liquid every three days. The brain tissue was transferred to solution C and soaked at room temperature for 72 hours. The liquid was changed every 24 hours. Under the condition of −20°C, the brain tissue was cut into 100um thick slices with a frozen microtome. Then the prepared working solution was used to dye the sections. The soaking sequence of slices was as follows: 50% ethanol for 4 minutes, 75% ethanol for 4 minutes, 95% ethanol for 4 minutes, absolute ethanol for 20 minutes and xylene for 20 minutes. Finally, the slices were sealed with a resin sealing agent and observed with a microscope.
5
Rapid Golgi Staining of Mouse Hippocampus
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Golgi staining was performed according to the manufacturer’s instructions of a FD Rapid Golgi staining kit (PK401, FD Neuro Technologies, United States). Mouse brains were harvested and immersed in the mixture of solution A and B (solution A:solution B = 1:1) for 2 weeks at room temperature in the dark. Then, the brain transferred to solution C for 4 days. The mixture and solution C were renewed within the first 24 h. Coronal brain sections (100 μm thick) of hippocampus were cut using a cryostat microtome (Leica, Wetzlar, Germany). The sections were further stained according to the manufacturer’s instructions. The stained slides were scanned using the Pannoramic scan digital slice scanner (3DHISTECH Ltd., Budapest, Hungary). The images were analyzed by ImageJ software. Secondary and third dendrites were sampled for spine density quantification.
6
Golgi Staining for Pyramidal Neuron Analysis
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Golgi staining was performed using the FD Rapid Golgi staining kit (PK401, FD NeuroTechnologies, United States) according to the manufacturer’s instructions. The brain samples were collected and immersed in a mixture of solutions A and B (1:1) in the dark at room temperature for 14 days. Thereafter, the samples were transferred to solution C in the dark for 5 days, embedded in optimal cutting temperature compound reagent, and frozen at − 80 ℃ for 24 h. The brains were cut into coronal Sects. (100 μm thick) using a cryostat microtome (Leica, Wetzlar, Germany). The staining procedure was performed following the manufacturer’s instructions. The slides were viewed using a light microscope with a 63 × oil-immersion objective lens (Olympus, FV1200). Pyramidal neurons in the CA1 region that were well-impregnated and clearly distinguishable from other neurons were analyzed. Five basal dendrite segments of 30 μm or longer were randomly selected from each pyramidal neuron. The dendritic spine density was analyzed using ImageJ.
7
Golgi-Cox Staining of Hippocampal Neurons
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Golgi-cox staining was completed with the FD rapid Golgi staining kit (FD NeuroTechnologies, MD) according to the manufacturer’s protocol. Briefly, animals were anesthetized and perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline. The brains were then dissected and stained following the manufacturer’s instructions. The slices were cut into a thickness of 200 μm with a Vibratome (Leica). Images of the hippocampi, neurons, and dendritic spines were acquired at × 20, × 400, and × 700 magnifications.
8
Rapid Golgi Staining of Brain Tissue
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Golgi staining was performed as previously described
28 (link) with an FD Rapid Golgi staining kit (FDNeuroTechnologies, Columbia, USA) according to the manufacturer's instructions. The kit consisted of solutions A to E. The brain was rapidly immersed in a mixture of solutions A and B and stored for 2 weeks (changed once within 24 hours) at room temperature in the dark to avoid decomposition. Then, the samples were transferred into solution C for at least 3 days. The brain was then cut into 100‐µm thick sections using a cryostat microtome (Leica). The sections were further stained according to the kit instructions. Images were observed and captured by an inverted microscope (IX73, Olympus) and analyzed by ImageJ v1.8 software.
28 (link) with an FD Rapid Golgi staining kit (FDNeuroTechnologies, Columbia, USA) according to the manufacturer's instructions. The kit consisted of solutions A to E. The brain was rapidly immersed in a mixture of solutions A and B and stored for 2 weeks (changed once within 24 hours) at room temperature in the dark to avoid decomposition. Then, the samples were transferred into solution C for at least 3 days. The brain was then cut into 100‐µm thick sections using a cryostat microtome (Leica). The sections were further stained according to the kit instructions. Images were observed and captured by an inverted microscope (IX73, Olympus) and analyzed by ImageJ v1.8 software.
9
Golgi-Cox Staining of TNC Tissue
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The FD Rapid Golgi Staining kit (FD Neuro Technologies, Inc) was used for Golgi-Cox staining. The TNC tissues were collected and immersed in the staining solution for 14 days at room temperature with dark light (replace the new dyeing solution after soaking for 48 h and then replace the new staining solution every 3 days). The TNC tissues were cryostat-cut by vibratome to obtain 100 μm thick sections, and sections were pasted to gelatin-coated slides. The staining process was carried out according to the manufacturer’s instructions. The images were acquired using an electron microscope (Nikon E100, Japan), and the panoramic image of the TNC tissue was obtained by the 3Dhistech Pannoramic Scan. For dendritic spines analysis, pyramidal neurons in the TNC region fully penetrated by the Golgi coloration were selected. Finally, the blinded researchers counted the number of spines on at least 3 apical dendritic segments of 20 μm in length to measure spine density using Image-pro Plus software. Each group contained three rats and five images per rat were investigated.
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