## Migration of Pigment Granules in Chromatophores from Cichlids (Amatitlania nigrofasciata): Week 2
* **IMPORTANT**: *You absolutely MUST wear closed-toe shoes and long pants to this lab. You will be handling toxins, and they are dissolved in an organic solvent that can diffuse through your skin. You will not be allowed to participate in the lab without proper footwear.*

* **REMINDERS**:
-You must wear a mask at all times while in the lab.
-You are expected to sanitize equipment and supplies upon entering and before leaving the lab.
-You are expected to clean up before you leave the lab. (Your lab bench/station should be left in the same state you found it on arrival: garbage thrown away, microscope, turned off, reagents put away, etc.)

*There is absolutely NO food or drink allowed in the labs at Colby College. Please leave any food or drink you bring with you in the hall outside of the lab.*
## Introduction
Today we will be examining the effects of epinephrine on pigment granule dispersal and aggreggation in chromatophores from Convict cichlids (*[Amatitlania nigrofasciata](https://www.fishbase.de/summary/Amatitlania-nigrofasciata.html)*), as a model system for studying cellular mechanisms controlling pigment distribution. The neurotransmitter epinephrine (adrenalin) has been reported to cause aggregation of pigment granules in some species of fish. The adrenergic receptor pathways activated by epinephrine are complex because there are different types of receptors and the receptors in turn can have their effects through different pathways. It has been reported that high intracellular calcium and low intracellular cAMP play a role in the response of some cell types to epinephrine. You can read more about cAMP and cell signaling in your textbook (Chapter 16).

## Objectives:
During this weeks lab you will continue to hone your skills in fish scale observation and experimental design/setup.
This week we will also be focusing on the Methods section. In future assignments you will work through how to put together the remaining sections of the poster so that by the end of the semester you are able to put together a complete scientific poster as a means of presenting the results of your project in lab this semester.

## Methods Section
The methods section of a scientific poster should include information pertaining to all of the basic parameters of your experiment including: the sample size, experimental conditions, timing of your experiment/steps of your experiment, the variables assessed, measurements used to assess your data and any analytical or statistical techniques that may have been used.
Your methods are in many ways a summary of your approach containing all of the critical information pertaining to your experimental approach. In this situation your methods helping the viewer to gain a better understanding of your approach to answering your question of interest. The goal is to provide your viewer with enough information that they will be able to walk away from your presentation with a basic understanding of the experiment(s) performed, but not so much that they would be able to go into have a back into the lab and repeat them.

Equally as important as the information being presented in your methods section of your scientific poster is that the presentation is visually striking and easy to follow. By this, I do not mean complicated. Rather that the information is presented in a easy to follow way that adds to the overall appearance of your poster. There is no single way of accomplishing this. You might consider including a schematic depiction of one or more of the steps of your procedure, pictures of the process, or, as we will practice today you might consider including a flow chart describing your procedure.

### Why a flow chart?

A flow chart is a diagram that depicts a process, system or set of relationships. Each unit or step represented in a flow chart is typically represented in a bounded box or shape and connected by arrows which indicate to the viewer how they should move through the chart. Steps may be linear, connected or branched to depict their relationships with one another.

Critically, flow charts meet all the basic criteria we have established for our methods section: they are a simple, logical means of presenting a often simplified series of steps that is also visually striking.

Example set up for a basic, unlabeled flow chart: (arrows indicate direction of movement between boxes/steps.)


## *As you work through the lab today and each week. Make sure the purpose for your experiments and your conclusions are clear in your notebooks. In addition, please include:*

1. A flow chart or complete protocol of the steps you actually followed in this lab.
2. An explicit description of your controls – what they were controlling for and the results and how this affected your interpretation of your experimental samples.
3. Don’t forget to record observations during the experiments. Even if things are not working like you would have hoped, it is always important to record all the data.
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## Basic Proceedure
We will walk through in class the basic proceedure for this lab which is a modification of the protocol we followed last week in lab and which I have included below.*
1. Place a scale in a depression slide with FRG.
2. Observe the scale for 5 minutes. You can use a cell phone camera to take pictures at regular intervals (try 30-60 seconds and/or try a movie).
3. Using a piece of blotting paper (*eg.* a Kimwipe (c)), wick away the FRG and rinse with perfusion buffer (PB) by filling the depression slide with it, wicking it away and refilling the depression slide with perfusion buffer. Work very quickly so that the scale does not dry out. Observe the scale as above.

****You should record this modified proceedure in your notes as it will be helpful in future weeks in lab.***

You will be given 3-4 scales which will be sufficient for this first set of experiments. The scales are submerged in fish Ringer’s (FRG), a salt solution that is 115mM NaCl, 2mM KCl, 1mM CaCl2, 0.1% glucose, 2mM Tris, pH 7. The other solution that you will use is perfusion buffer, with the detergent digitonin for permeabilization of the membrane: 33mM potassium acetate, 30mM HEPES, 10mM EGTA, 5mM MgSO4, 0.5mM EDTA, 2.5% polyethylene glycol, pH 7.4.

Additional reagents available for this weeks for pilot experiments:
1. 0.1 mM epinephrine in fish Ringers with digitonin
2. 0.1 mM epinephrine in perfusion buffer with digitonin
3. fish Ringers with digitonin
4. perfusion buffer with digitonin

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**In your notebook, begin by describing the practice experiment you are doing today:**

*What are the hypotheses that you are testing in this experiment?*

*Which treatment condition(s) are your control?*

*Which treatment condition(s) are your experimental?*

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### Let’s practice making a flow chart:

One way to converting a working protocol that you might use in the lab into a more visual depiction of the methods used in conducting an experiment which might be included on a Scientific Poster is to make a flow chart.

**In a flow chart:**

• Each step in your protocol should be depicted in its own box.
• Connect the steps using arrows so each step is connected to the next.
• If you repeat a step or group of steps, then connect that box to the first step that is to be repeated.
• If you repeat something more than once, be sure to indicate that on your flow chart as well.

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**Make a flow chart for the experiment we are performing today in lab and record that in your notebook.**

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**Now consider whether there are any steps that you have included in your flow chart that you might leave out on a scientific poster.**

*If so, which steps are these? Why?*

*If not? Why not?*

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Is there any additional information that you would like to/need to include on your flow chart in order to fully describe your experimental approach?

If so, what? How might you include this data/information with your flow chart?

Is there any information that you feel is necessary to include, but you are unable to fit into your flow chart?

Could you fit this information in by depicting your methods in a different way? If so, how?

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**Make all the adjustments you have indicated above and record that in your notebook.**

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Your assignment this week is to come up with a plan for your experiments over the coming weeks. By the end of this week (Friday at midnight) you will need to tell me what reagents you will require for your experiments.
To facilitate this, please complete the following:

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Hypothesis, Experimental Design, Reagent requests

**Names of Group Members:**

**1. Hypothesis**

a. Briefly state the null hypothesis you will be testing in weeks 3 and 4 of lab.

b. What are your experimental predictions based on the null hypothesis that you are testing?

c. Given your hypothesis, briefly state give one alternative null hypothesis.

d. What are your experimental predictions based on the alternative null hypothesis you have provided?

**2. Experimental Design**

a. Briefly describe the experiment(s) you plan to complete week 3 and 4.

b. What are your negative controls for this/these experiment(s)?

c. Are you planning to include a positive control in your experiments? Why/why not?

d. Will your planned experiments have generated replicate data? Why or why not?

e. Below please include a flowchart indicating the ordered set of steps necessary to complete the experiments you have described. Please include all planned starting and stopping points for your experiments (e.g. what you will do each week).

**3. Materials**

Please list the reagents you will need to conduct your experiment(s). Include the name of the reagents and the doses you will use.

Double-check your experimental design. Be sure you have included everything you will need for both your control and experimental conditions and for both weeks of experiments. Some reagents will need to be ordered, this takes time, so last minute adjustments may not be possible.

**Once completed, please submit this information to me via the course Moodle site.**