TITLE: A single guide to impregnate samples with Golgi-Cox solution within 24hr and represent results with a set of algorithm
AUTHORS: Avishek Roy, Binney Sharma

[DESCRIPTION]
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. 

[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.   

[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. 

[STEPS]
SECTION: Transcardial perfusion
11. Perfusion setup was filled with 0.9 % NaCl (saline) 2-4 °C  and the flow rate was set at a rate of 3ml/min 

SECTION: Transcardial perfusion
12. 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
SECTION: Transcardial perfusion
13. Finally, the saline needle was introduced to the left ventricle and then the right auricle was incised to break the close loop
SECTION: Brain isolation
14. After the completion of perfusion process, animals can be decapitated to isolate the complete brain
SECTION: Brain isolation
14.1. Lateral incision was made by occipital bone, followed by I-incision through sagittal suture
SECTION: Brain isolation
14.2. Finally, the nasal bone was broken to peel off the skull bone in order to isolate brain
SECTION: Preparation of coronal chunks
15.   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)
 



SECTION: Preparation of developing solutions
2. For developing the impregnation colour we have used following reagents:
  
  
  

SECTION: Preparation of developing solutions
2.3. 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) 
SECTION: Preparation of developing solutions
2.2. 2 part of Ammonium hydroxyde was mixe with 1 part of MiliQ water to prepare 3:1 (V/V) ammonium hydroxyde
SECTION: Preparation of developing solutions
2.1. A 5% (W/V) solution of sodium thiosulfate was prepared mixing 100ml of MiliQ water in 5gm of sodium thiosulfate
SECTION: Slide cleaning and coating
3. In order to make the section stick to the slides first we have kept frosted micro slides (Bluestar, 75mm x 25mm) in    18.5 % volume for   

SECTION: Slide cleaning and coating
4. HCl was discarded and replaced with running tap water for 2-3hr 
SECTION: Slide cleaning and coating
5. Running tap water was discarded and replaced with 50 % (v/v) of admixture of      and kept for 2-3hr

SECTION: Slide cleaning and coating
6. Again the admixture was replaced with running tap water and slides were kept for 1-2hr
SECTION: Slide cleaning and coating
7. 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

SECTION: Slide cleaning and coating
8. A solution of 3 % (v/v)     was prepared mixing gelatin in MiliQ water (eg. 3gm in 100ml of MiliQ) 


SECTION: Slide cleaning and coating
8.1. Cleaned slides were incubated in the 3% Gelatin solution at 40 °C  inside incubator (BIOOCN India, India) for 10 min 

SECTION: Preparation of Golgi-Cox solution
1. Golgi-Cox solution was prepared using          

 

SECTION: Preparation of Golgi-Cox solution
1.1. First, we have prepared 5 % solution of all three aforementioned salts from 15gm of salts dissolving into 300ml of MiliQ water

SECTION: Preparation of Golgi-Cox solution
1.2. Next, mercury(II) chloride and potassium-di-chromate was mixed at  1:1 ratio (:)  

SECTION: Preparation of Golgi-Cox solution
1.3. Potassium chromate was added at 4 % (v/v) to the previous mixture 

SECTION: Anaesthesia
9. Rats were treated with lethal dose of sodium thiopentone (150mg/kg of BW) through intra-peritoneal route 
SECTION: Anaesthesia
10. Level of anaesthesia was checked though paw-pressor test
SECTION: Impregnation step
16. 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.  
SECTION: Impregnation step
16.1. 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 1440 min  (see fig. 1.E-F in attachment)

SECTION: Block preparation
17. Wash brain chunks incubated with Golgi-Cox solution in 30 Mass / % volume sucrose in MiliQ at Room temperature for 10 min 

SECTION: Block preparation
18. Prepare 3 %    in MiliQ and replace the 30 %  sucrose solution with the same pouring gently into a customized mold from 50ml falcon tubes (see attachment; Fig. 3B in attachment)
 
 These can be preserved at 4 °C for 20160 min  when sealed with parafilm till vibrotomy

SECTION: Sectioning and transferring them to slides
19.   thick coronal sections were prepared with vibratome in a solution of 6 %  sucrose made in MiliQ
 



SECTION: Sectioning and transferring them to slides
20. Once sections are prepared they were immediately transferred onto the pre-coated frosted glass slides          
SECTION: Sectioning and transferring them to slides
21. Finally, extra solutions were wiped with a gentle pressure of palm with a tissue wet in 6 %  sucrose 

 

SECTION: Sectioning and transferring them to slides
22. These slides were then kept in a humidified chamber and again placed into a incubator at 37 °C  for 1440 min 
 


SECTION: Developing and mounting
23. Sections were hydrated with MiliQ water at Room temperature for 5 min 

SECTION: Developing and mounting
24. Transferred to 50 %  ethanol at Room temperature for 5 min 

SECTION: Developing and mounting
25. Transferred to 3:1 ratio (:) ammonium solution at Room temperature  for 10 min 

SECTION: Developing and mounting
26. Rinsed in MiliQ water at Room temperature  for 5 min 

SECTION: Developing and mounting
27. Transferred to 5 %  sodium thiosulfate solution at Room temperature  for 12 min 

SECTION: Developing and mounting
28. Rinsed again with MiliQ water at Room temperature  for 2 min 

SECTION: Developing and mounting
29. Sections were dehydrated with graded alcohol (70, 95, 100: 5 min each)
SECTION: Developing and mounting
30. Transferred to Xylene at Room temperature  for 10 min 

 

SECTION: Developing and mounting
31. Now sections can be mounted in a coverslip with DPX 
SECTION: Imaging and tracing
32. After one day at DPX slides can be imaged in bright-field microscope.
SECTION: Imaging and tracing
32.1. 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 120 min  for a complete tracing of neurons like pyramidal cells  in hippocampus. 
   
 




SECTION: Imaging and tracing
32.2. The same thing can be achieved by making a stack out of single plane images at ≤   thickness and then tracing using Matlab or Image J plugins r even with some other standalone softwares:

 
    
 

 





SECTION: Data extraction
33. 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
 
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 


 

Or we can upload ASC file to the "Patchview' / NeuroM software and can perform Sholl analysis
 
Or even in NeuromorphoVis

 

This process should not take more than 5 min  per neuron traced
 

SECTION: Visualization of neuron
34. 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: 

 

 
This can be done with few clicks in the software GUI 
   
  

SECTION: Analysis and its types
35. 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