May 30, 2023
Version 2
A single guide to impregnate samples with Golgi-Cox solution within 24hr and represent results with a set of algorithm V.2
- 1All India Institute of medical Sciences, New Delhi-110029, India;
- 2Interdisciplinary Institute for Neuroscience, UMR-5297, University of Bordeaux, Bordeaux Cedex, France
Protocol Citation: Avishek Roy, Binney Sharma 2023. A single guide to impregnate samples with Golgi-Cox solution within 24hr and represent results with a set of algorithm. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jdrolo5/v2Version created by Avishek Roy
Manuscript citation:
Roy, A., Sharma, S., Nag, T.C.et al.Cognitive Dysfunction and Anxiety Resulting from Synaptic Downscaling, Hippocampal Atrophy, and Ventricular Enlargement with Intracerebroventricular Streptozotocin Injection in Male Wistar Rats.Neurotox Res40, 2179–2202 (2022). https://doi.org/10.1007/s12640-022-00563-x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 30, 2023
Last Modified: May 30, 2023
Protocol Integer ID: 82647
Keywords: Golgi-Cox staining, One day impregnation, Neuronal morphometry
Funders Acknowledgement:
Indian Council for Medical Research
Grant ID: IR-594
Abstract
Golgi-Cox staining is one of the old but relevant histological technique to identify neurons in superficial/ deep brain structures. The goal of this staining is to accurately perform morphometric analysis on the desired neurons in health and diseases. Due to the origin of its own there are different variations of the protocol itself. Majority of them take at least one to two week to have impregnation of the stain into the tissue/ cells. This is due to the physical property of the stain and lipophilic nature of the central nervous system. Therefore to enhance the diffusion of the stain into the brain samples, we have came up with a modification of only one protocol out there by Ranjan and his colleagues. Where we have decreased the thickness of the brain sample to 5mm from 25±1mm (for rat brain) and incubated the sample with Golgi-Cox solution at 37°C in order to reduce the path of travelling and simultaneously help the diffusion process at physiological temperature. The results showed a significant amount of impregnation of the Golgi-Cox solution into deep brain structures viz. hypothalamus, hippocampus within a timespan of 24 hour. This reduces the labour, time and enhance the efficiency of the impregnation process enabling a better image to analyse further. The overall goal of this protocol is to help experimenter focusing on the analysis of the morphometric data as well as complementing it with other relevant functional data by reducing the time to stain samples enhancing efficacy. Finally, we hope that this modified protocol will not only help researchers in field of neuroscience to perform the technique with ease but also help them to represent their result in the best/ unique way using different algorithm and softwares mentioned in the protocol.
Attachments
Attachment to the pr...
32.6MB
Guidelines
This protocol is meant to reduce the time and increase the efficiency of the Golgi-Cox staining in central nervous tissue. Due to the reason that this protocol is modified from the available literatures keeping in mind the basic principles of diffusion of a metal i.e. physical properties viz. temperature, thickness etc. This protocol may vary a little in sample preparation steps. We have got robust staining in every batch of samples we have stained using the same protocol. There is also few literature where use of penetrating agents viz. SDZ, triton X has been used alongside the same protocol and showed no further improvement. However, we have not tried ,manipulating that, however comparison with the regular 07 day protocol with change of solution at one week interval showed less efficiency in staining the deep structures. Availability this protocol will help researcher to focus on more critical analysis and presentation of the morphological data one can get from this technique.
We recommend to use fresh solutions with a filtration at least at an interval of 3-4weeks. And use of glassware/ plasticware during the whole procedure to avoid any kind metallic reaction. Further, keep the sections in dark especially in the slicing, humid chamber, and even during developing step as possible.
Materials
Animals step 9
Vibratome step 19
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641Step 5
Mercury(II) chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1136-100GStep 1
Agar agarMerck MilliporeSigma (Sigma-Aldrich)Catalog #05038-500GStep 18
Sodium thiosulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #72049-250GStep 2
Ammonium hydroxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #05002-1LStep 2
Ethanol Merck Millipore (EMD Millipore)Catalog #100983Step 2
Hydrogen chloride solution (HCl 1M)Merck MilliporeSigma (Sigma-Aldrich)Step 3
Ethanol PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #493511Step 5
500g Gelatin (Reagent Grade)G-BiosciencesCatalog #RC-053Step 8
Protocol materials
Agar agarMerck MilliporeSigma (Sigma-Aldrich)Catalog #05038-500G
Step 18
500g Gelatin (Reagent Grade)G-BiosciencesCatalog #RC-053
Step 8
Mercury(II) chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1136-100G
Step 1
Sodium thiosulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #72049-250G
Step 2
Ammonium hydroxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #05002-1L
Step 2
Ethanol Merck Millipore (EMD Millipore)Catalog #100983
Step 2
Hydrogen chloride solution (HCl 1M)Merck MilliporeSigma (Sigma-Aldrich)
Step 3
Ethanol PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #493511
Step 5
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
Step 5
Potassium chromateMerck MilliporeSigma (Sigma-Aldrich)Catalog #12249-100G
Step 1
Potassium di-chromateMerck MilliporeSigma (Sigma-Aldrich)Catalog #207802-100G
Step 1
Safety warnings
This protocol includes use of heavy metals, paraformaldehyde and other potential carcinogens hence the researcher is suggested to use proper safety gears, and use of biosafety hood whenever possible.
Ethics statement
This study was performed under ethical clearance from Institutional Animal Ethical Committee (IAEC), All India Institute of Medical Sciences, New Delhi, which is under Committee for the Purpose of Control and Supervision of Animal experiments CPSCEA, India. Vide no. 937/IAEC/PhD-2016.
Before start
Before start one should have basic knowledge of central nervous system and have experience on microscopic experiments and histological experiments. However, we have tried to prepare this protocol keeping a larger readers in mind, and the motto of this protocol is to get sample stained on the first trial with just a knowledge/ experience in wet lab. Further, as the main criticality of the technique lies on the analysis of the results one can extract from the morphometry of the stained neurons, therefore a knowledge on the neuronal architecture specifically to the brain region you are interested with is an advantage. We have kept the minimum duration of the incubation/ impregnation to be 24 hr keeping in mind that once you go to >2days you might have more stained neurons and their branches, but you will loose the branch of a specific cell you are tracing. This problem happens when one neurite is being masked by another neighbouring neuron/ neurite.
Preparation of Golgi-Cox solution
Preparation of Golgi-Cox solution
25m
25m
Golgi-Cox solution was prepared using Mercury(II) chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1136-100G Potassium chromateMerck MilliporeSigma (Sigma-Aldrich)Catalog #12249-100G Potassium di-chromateMerck MilliporeSigma (Sigma-Aldrich)Catalog #207802-100G
Safety information
All these reagents are toxic/ carcinogenic therefore use of these reagents should be conducted with proper safety gears:
1. Prepare solution under safety hood
2. Use gloves +/ glasses as possible
3. Due to the metal in this stain avoid using any other equipment except it is made of plastic/ glass. Not even metal forceps
25m
First, we have prepared 5 undetermined W/V solution of all three aforementioned salts from 15gm of salts dissolving into 300ml of MiliQ water
15m
Next, mercury(II) chloride and potassium-di-chromate was mixed at 1:1 Mass Percent V/V
5m
Potassium chromate was added at 4 % (v/v) to the previous mixture
5m
Preparation of developing solutions
Preparation of developing solutions
17m
17m
For developing the impregnation colour we have used following reagents:
Sodium thiosulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #72049-250G
Ammonium hydroxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #05002-1L
Ethanol Merck Millipore (EMD Millipore)Catalog #100983
17m
A 5% (W/V) solution of sodium thiosulfate was prepared mixing 100ml of MiliQ water in 5gm of sodium thiosulfate
5m
2 part of Ammonium hydroxyde was mixe with 1 part of MiliQ water to prepare 3:1 (V/V) ammonium hydroxyde
2m
In order to make ascending order of alcohol concentration (50%, 75%, 95%, 100%) we have mixed MiliQ water with Ethanol in the aforementioned ratio (V/V)
10m
In order to make the section stick to the slides first we have kept frosted micro slides (Bluestar, 75mm x 25mm) in Hydrogen chloride solution (HCl 1M)Merck MilliporeSigma (Sigma-Aldrich) 18.5 % volume in MiliQ water for Overnight in the staining trough
12h
Etching process38 steps
This step is done to increase the surface area, which will help sections to stick to the slide while staining
HCl was discarded and replaced with running tap water for 2-3hr
3h
Running tap water was discarded and replaced with 50 % (v/v) of admixture of Ethanol PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #493511 MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641 and kept for 2-3hr
3h
Again the admixture was replaced with running tap water and slides were kept for 1-2hr
2h
Removal of any lipophilic substances35 steps
This step is important to remove any oily substances/ grease from the surfaces of the slides
Finally tap water was replaced with MiliQ water and slides were dried in slide racks inside incubator (BIOOCN India, India) at warmer 60-70 °C
Now the slides are ready to coat
30m
A solution of 3 % (v/v) 500g Gelatin (Reagent Grade)G-BiosciencesCatalog #RC-053 was prepared mixing gelatin in MiliQ water (eg. 3gm in 100ml of MiliQ)
Cleaned slides were incubated in the 3% Gelatin solution at 40 °C inside incubator (BIOOCN India, India) for 00:10:00
10m
Anaesthesia
Anaesthesia
5m 50s
5m 50s
Rats were treated with lethal dose of sodium thiopentone (150mg/kg of BW) through intra-peritoneal route
Level of anaesthesia was checked though paw-pressor test
CO2 Asphyxiation30 steps
One can opt for euthanasia in CO2 chamber by treatment of >60% CO2. And at the end level of anaesthsia could be tested as in step 3
Perfusion setup was filled with 0.9 undetermined W/V NaCl (saline) 2-4 °C and the flow rate was set at a rate of 3ml/min
5m
Minimum volume of saline to be perfused29 steps
25-35 ml of ice cold saline can be perfused or else one can perfuse till the lungs and kidneys get white. This indicates saturation of the fluid at pulmonary as well as aortic circuit respectively.
Rats were placed onto their back and heart was made visible by opening cardiac envelop followed by an access to plural cavity through incisions through diaphragm
3m
Finally, the saline needle was introduced to the left ventricle and then the right auricle was incised to break the close loop
40s
Brain isolation
Brain isolation
2m 20s
2m 20s
After the completion of perfusion process, animals can be decapitated to isolate the complete brain
20s
Lateral incision was made by occipital bone, followed by I-incision through sagittal suture
1m
Finally, the nasal bone was broken to peel off the skull bone in order to isolate brain
1m
Preparation of coronal chunks
Preparation of coronal chunks
50s
50s
5 mm coronal chunk of the brain from anterior to posterior was cut using the brain matrice (51388, Stoelting Co., USA) (see fig. 1.C-D in attachment)
Equipment
Brain Matrices
NAME
Crude tissue slicer
TYPE
Stoelting CO. (USA)
BRAND
51388
SKU
LINK
1.0 mm thickness coronal slices
SPECIFICATIONS
50s
Impregnation step
Impregnation step
1d
1d
Immerse your sample (brain chunks viz. frontal lobe, cerebellum, spinal cord etc.) in the filtered Golgi-Cox solution as prepared in step 1. Keep in mind to use Golgi-Cox at 10X the volume of the sample.
Keep the preparation in amber colour bottle (or use aluminium foil to wrap in any glass/ plastic bottle available) at 37±2 °C for minimum 24:00:00 (see fig. 1.E-F in attachment)
1d
Block preparation
Block preparation
10m
10m
Wash brain chunks incubated with Golgi-Cox solution in 30 Mass / % volume sucrose in MiliQ at Room temperature for 00:10:00
10m
Prepare 3 undetermined Agar agarMerck MilliporeSigma (Sigma-Aldrich)Catalog #05038-500G in MiliQ and replace the 30 undetermined W/V sucrose solution with the same pouring gently into a customized mold from 50ml falcon tubes (see attachment; Fig. 3B in attachment)
Equipment
Corning® 50 mL centrifuge tubes
NAME
conical bottom tube
TYPE
Corning 430829
BRAND
CLS430829-500EA
SKU
These can be preserved at 4 °C for 336:00:00 when sealed with parafilm till vibrotomy
2w
Sectioning and transferring them to slides
Sectioning and transferring them to slides
1d 0h 36m
1d 0h 36m
300 µm thick coronal sections were prepared with vibratome in a solution of 6 undetermined sucrose made in MiliQ
Equipment
Vibrotome
NAME
Slicing sections at higher thickness
TYPE
Leica
BRAND
VT1000 S
SKU
30m
Once sections are prepared they were immediately transferred onto the pre-coated frosted glass slides
5m
Finally, extra solutions were wiped with a gentle pressure of palm with a tissue wet in 6 undetermined sucrose
Note
Put gentle pressure at a specific angle (90 degree) with the wet tissue paper. This step not only help you get rid of extra sucrose/ cutting solution but also stable the sections which will help you mounting them at the last step of staining.
Keep in mind not to p[ut pressure at different angle at a same time by moving the palm over the slide. If you do so, then there is a risk of loosing the orientation of the sections as well as making an irreversible impression on the section
1m
These slides were then kept in a humidified chamber and again placed into a incubator at 37 °C for 24:00:00
Note
This step is required to fix the sections onto the slides. If not carried out then there is a high risk of loosing the sections during developing step
1d
Developing and mounting
Developing and mounting
1h 5m
1h 5m
Sections were hydrated with MiliQ water at Room temperature for 00:05:00
5m
Transferred to 50 undetermined ethanol at Room temperature for 00:05:00
5m
Transferred to 3:1 Mass Percent ammonium solution at Room temperature for 00:10:00
10m
Rinsed in MiliQ water at Room temperature for 00:05:00
5m
Transferred to 5 undetermined sodium thiosulfate solution at Room temperature for 00:12:00
12m
Rinsed again with MiliQ water at Room temperature for 00:02:00
2m
Sections were dehydrated with graded alcohol (70, 95, 100: 5 min each)
15m
Transferred to Xylene at Room temperature for 00:10:00
Note
Sections can stay in the xylene solution until they are mounted however more than 10-12 minute in our hand causes a significant tissue brittle, breakage at spaces
10m
Now sections can be mounted in a coverslip with DPX
1m
Imaging and tracing
Imaging and tracing
2h
2h
After one day at DPX slides can be imaged in bright-field microscope.
While imaging at higher magnification (preferably at 100X with oil-immersion) you can trace your neuron with the specific segmentation annotation i.e. giving name to the traces viz. soma, basal dendrite, apical dendrite etc. Further, in the same time you can also tag your neurites with specific type of spines (viz. mushroom, thin, stubby etc.) this process can take 02:00:00 for a complete tracing of neurons like pyramidal cells in hippocampus.
Comparative images with regular 7 days incubation at room temperature (Ai-ii); and at physiological temperature with thin slices (Bi-ii).
Note
There are different softwares available for neurite tracing (see fig. 4 A-B in attachment)
Software
Neurolucida
NAME
Windows 10, 64-bit
OS
MBF BioSciences
DEVELOPER
2h
The same thing can be achieved by making a stack out of single plane images at ≤ 1 µm thickness and then tracing using Matlab or Image J plugins r even with some other standalone softwares:
Software
Simple Neurite tracer
NAME
Tiago Ferreira
DEVELOPER
SOURCE LINK
Tracing with SNT plugin in Image J/ FIJI; in this scheme of images you can follow directly to perform tracing using SNT (neuroanatomy plugin) from step1-12. This is also quite easy protocol we have used a premade stack of image kindly provided by
Mr. Ignacio Javier Novoa,Brain Plasticity and Neurorehabilitation Laboratory (BPNL, https://www.muthaiahlab.com/).
Software
ShuTu
NAME
Windows/ Mac/ Ubuntu
OS
Dezhe Jin
DEVELOPER
Software
Neurite Tracing With Object Process
NAME
Matlab
OS
Shreetama Basu
DEVELOPER
Data extraction
Data extraction
5m
5m
Once the neurons are traced it is ready to get the data out of it. Traced neurons can be saved in different file formats:
1. dat
2. ASCII
3. SWC
However, we can change the format at anytime with a NLMorphology converter/ Neuroland viewer
Software
NLMorphology Converter
NAME
Next, data can be directly extracted from SNT plugin/ neuroanatomy package by doing Sholl analysis function or you can go for Neurolucida Explorer for the same (MBF Biosciences, USA)
Even we can do the same in Shutu/ NeuTube
Software
ShuTU/NeuTube
NAME
Dezhe Jin
DEVELOPER
Or we can upload ASC file to the "Patchview' / NeuroM software and can perform Sholl analysis
Or even in NeuromorphoVis
Software
NeuroMorphoVis
NAME
BlueBrain Project
DEVELOPER
This process should not take more than 00:05:00 per neuron traced
5m
Visualization of neuron
Visualization of neuron
1m
1m
Visualization of the neuron is secondary to the analysis of data extracted from the tracings.
However, this is important in the sense that you can represent and compare between treatments/ cases. For the same Neurolucida/ Imaris already will do the job however if you want work with free/ open source softwares then it can be done in following softwares:
Software
NeuroMorphoVis
NAME
BlueBrain Project
DEVELOPER
Software
Neuronize
NAME
Windows
OS
Visualization & Graphics Lab
DEVELOPER
This can be done with few clicks in the software GUI
Reconstructed and processed images of 200um thick sections from control animal hippocampus n]in regular ways. CA1 pyramidal neuron; 400X magnified image of dendritic complexity (a); 1000X magnified image of tuft dendrite spines ((b); 1000X magnified image of apical dendrite spines (c). Scale bar=50µm
Ways to represent results apart from morphometric results; convex hull representation of a CA3 neuron (A); fan diagram of a CA1 neuron (B); hypothalamic neuron traced and rendered with Neuronize(C); 3D surface plot of a CA1 neuron in 3D view tool in Image J (D).
1m
Analysis and its types
Analysis and its types
For morphometric analysis of neurons we generally perform following type of analysis:
1. Spine density calculation (which can be extracted from the tagged spine during tracing)
2. Type of spine (this approach is good when your research question is more restricted to the types of spine viz. mushroom-shaped spine as this is site for glutamatergic synapse)
3. Sholl analysis where you perform one variable (length/ intersection) versus the distance from soma
4. Branch structure analysis where you mainly perform the various parameters related to branch viz. number of terminal branches, turtuosity, branch order etc.
5. Convex hull analysis where one can measure the volume of the neurite or soma
Protocol references
Rutledge LT, Duncan J, Beatty N. A study of pyramidal cell axon collaterals in intact and partially isolated adult cerebral cortex. Brain Res. 1969 Nov;16(1):15-22. doi: 10.1016/0006-8993(69)90082-1. PMID: 4186864.
Ranjan, A., and Mallick, B. N. (2010). A modified method for consistent and reliable Golgi-cox staining in significantly reduced time. Front. Neurol. 1:157. doi: 10.3389/fneur.2010.00157
Zaqout S and Kaindl AM (2016) Golgi-Cox Staining Step by Step. Front. Neuroanat. 10:38. doi: 10.3389/fnana.2016.00038