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BNS tissue sectioning :

1. Open valve to CO2 tank and cool stage until completely frozen 

2. Cut Straight across cerebellum so brain can Stay level and upright

3. Apply 1% ethanol to frozen stage as the base

4. When ethanol is halfway frozen add a layer of 30% sucrose to the base

5. When sucrose is halfway frozen, place brain so that the dorsal side will face towards the blade

6. Add more sucrose around the tissue and let it freeze

7. Manually raise/lower the Stage so that the tissue is below where the blade will run

8. Place blade in its designated space and tighten screws to hold the blade in place

9. Obtain a brush to handle the tissue and sectioned dish full of distilled water to place tissue

10. Begin cutting tissue into sections of the desired thickness (50 microns) and fully extend cutting arm for automatic adjustments

11. When done cutting, remove blade, wash with soap and water, then dry. Coat blade in oil and store until next use.

12. For mounting, place one section in dish of water, float to the top and place slide under tissue in water

13. Using a brush guide tissue to its spot on the slide 

14. When done mounting allow tissue to dry completely before staining

Nissil Stain Video:

1. 6 containers will be needed

   1. Distilled water

   2. 70% Alcohol

   3. Absolute alcohol

   4. Clear Safe

   5. Violet Stain

   6. Acid Alcohol (will be used in step 10)

2. Place each slide in its own section in the distilled water 

• Leave in for 2 minutes (this is to get rid of sucrose)

3. Then place slides in the 70% alcohol 

• Leave in for 2-4 minutes (4 minutes is better 

• This is used to start the dehydrating process 

• The alcohol phases have to happen because distilled water and clear safe cant mix

4. Place into absolute alcohol

• Leave in for 5 minutes (also part of dehydration phase)

5. Place into Clear Safe solution 

• Leave in for 5 minutes (if the mixture gets cloudy, it means the slides have to go back into the alcohol because they are not dehydrated enough)

• This will make the brain sections clear because it gets rid of fat

• Since this is water based, the sections have to back through the alcohols before the stain

6. Back into absolute alcohol (4 minutes)

7. Back into 70% alcohol (4 minutes)

8. Back into distilled water (2 minutes)

9. Then place the slides into the stain

• Leave in the stain for 20 minutes (usually 10 minutes but it is found that leaving it in the stain for longer is better)

• While waiting for stain place the Acid Alcohol in between the distilled water and the 70% alcohol containers

10. When the slides are done in the stain place back into the water (have waste bucket near)

• Leave in for 4 minutes, when the water starts to significantly change color pour it out and refill

11. Place the slides in the Alcohol Acid 

• Leave it in for 2-4 minutes and change the solution again when it starts changing color

12. Place into the 70% alcohol and watch it

13. Place them into the absolute alcohol for several minutes keeping an eye on them 

14. Then place in clear safe and leave there while preparing cover slides

Coverslides:

• Get same number of cover slides as needed for how many slides you have 

• Use paramount to stick the cover slides to the slide

• Take the slide directly  from the Clear safe solution for this process

• Ensuring the pieces of brain are facing up, tilt the slide away from you

• Using the end of a q-tip, take paramount and place it along the bottom of the slide (the part of the slide facing away from you)

• Slide the cover slide from the bottom near the paramount to onto the slide

• Tap the sides on the table a bit to make sure the slides are aligned 

• Repeat with the rest of the slides and then allow them to dry

I used duration testing to record the amount of time this person spent playing with her hair. The reason I used duration testing is because I figured that she would be playing with her hair for longer periods of time. I started recording every time she started playing with her hair and stopped the timer when she stopped. Then I quickly restarted the timer to track how long she did not play with her hair. I noticed a pattern that when she was doing two things at once, such as writing down notes AND looking at her computer she would not play with her hair, even if one hand was open. However, when she was only doing one activity, such as writing she would play with her hair. I studied her from 11:46 am - 12:46 pm. 

Results of duration testing: 

• Time spent playing with her hair: 37 minutes 

• Time spent not playing with hair: 17 minutes 

The missing time of about 6 minutes is observer error on my part and is most likely lost time between tests. Although duration has its strengths, for example, it is easier than the other tests because you can record activites for long periods of time with out having to make every time an activity happens. This form of testing also has its weaknesses, for example, losing time between tests if you were not fast enough to restart the clock, and it can be inefficient for certain tests. 

Some other observations: 

• While talking she did not play with her hair

• When confused or trying to figure something out, she did not play with her hair

• Depending on the activity the area of her hair which she would play with changed:

  • While looking at her computer/phone she would mainly touch the roots of her hair

  • While writing things down she was mainly touching the ends of her hair

  • This could be because of the different positions her head was in while doing different activities

• I researched a bit about why people play with their hair and most of the results is that it is either a calming/soothing activity for them, or it has just generally become a mindless habit in their life

After watching all of the videos I made a scoring system reflecting the anxiety levels of the mouse. The scoring system:

Hands on the wall -3 

In the middle +3

3 inches from wall +2

Touching wall with body -1

Nose facing wall -2

Body length away from wall +4

Stopped in middle +3

The positive numbers represented social behavior and the negative numbers represented anxious behavior. I collected all the data based on the numbers and added them together at the end. If the mouse got an overall positive number, it means that they were more social and if they got an overall negative number, it means they were more anxious. 

https://www.sciencedirect.com/science/article/abs/pii/S0166432818302882?via%3Dihub

The author is Allie Holschbach. Allie Holschbach is an associate professor of neuroscience at NSU Florida. She is very far into her career as she is teaching at a University. She focused her research on testosterone, pair bonding, and parental behavior in mice. Then she did some breakthrough research on postpartum depression in mice. The bulk of her research is due to serotonin receptors, especially the effect of stress on serotonin in both male in females. 

https://www.sciencedirect.com/science/article/abs/pii/S0031938418303792#:~:text=Because%20activity%20of%20the%20neuropeptide,nor%20was%20there%20an%20interaction).

• This is a study based on what affects the lactation in the mother, if it is the postpartum changes, or if the litter age affects this. 

https://pubmed.ncbi.nlm.nih.gov/22101260/

• This is a study that shows that reproductive success depends on the female's assessment of the male mouse.