Overview

Introduction

On the pages listed below you can find most common protocols used in the lab as well as a list of lab duties along with the names of the responsible persons. Please, contact the responsible persons if you have any question, suggestions or troubles.

Axolotl

Feeding

Artemia (Brine shrimp eggs)

Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours.

Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.

Artemia Hatching Preparation: Protocol for 15 Ltr.

Switch on the bubbler.
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.
Switch on the heating. And make sure that the light on the top is on ☺ Add 50-60 ml full of dried artemia (Stored at 40C) to this.

Hatching preparation

• The following protocol is considered for 15 l solution.
• Switch on the bubbler.
• Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.
• Switch on the heating. And make sure that the light on the top is on.
• Add 50-60 ml full of dried artemia (Stored at 40C) to this.

Keep it like this for 2 days with aeration. And >200 Lux of light.
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.
During these two days temperature can be 18°-20°C.
Make sure hatching container is not getting clogged during these two days.

Lab duties

Chemicals

• Antibiotics
• Benzocaine
• Fibronectin
• Gelatine
• Liquid nitrogen
• Primary antibodies
• Secondary antibodies
• Serum immunostaining

Hardware

• Autoclave
• Cryostat
• Dissecting microscopes
• Leica confocal microscope
• Vacuum pumps
• Waterbath

General

• Ordering common stuff
• Heat-shock competent cells

Protocols

Cell culture

• Blastema cells
• Baculovirus titration
• Myoblasts electroporation
• Adhesive cell culture
• Electroporation of blastema cells
• Electroporation of neural epithelia cells (axolotl spinal cord)
• mESC culture/Cyst formation
• Neurosphere culture

Histology

Molecular biology

Booking calendars

Since some pieces of hardware are used intensively it is necessary to book them in advance. The links to the corresponding booking calendars are listed below.

• Fluorescence microscopes
  • Olympus SZX 16
  • New Olympus SZX 16
  • Olympus OVK
  • Axio Observer
• Microinjectors and electroporators
  • Electroporator
  • 2nd electroporator
  • Microinjector
  • Stereoscope
• Confocal microscopes
  • Leica LSM
• Cryostats
  • Cryostat 1
  • Cryostat 2
• Microtome
  • Microtome

Transgenic lines

Axolotl (A.mexicanum)

Name	Construct	Method of transgenesis	Author	Tissue	Image(s)	Remarks
PM28	Prrx1-TFP-NLS-T2A-mERt-Cre-ERt	Sce1	Prayag Murawala	Limb bud and limb blastema	Error creating thumbnail: File missing Limb bud expression	Germ-line transmitted
PM33	Col1A2-TFP-NLS-T2A-mERt-Cre-ERt	Sce1	Prayag Murawala	Bone and fibroblast	Error creating thumbnail: File missing Bone expression Error creating thumbnail: File missing Bone expression in limb bud	Germ-line transmitted

Digital data

Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on. Select a category from the list below in order to view or download the data.

Theses

Title	Summary	Author	Link
Test	Test	John Doe	tba

Databases

• Axolotl EST database contains several ESTs and also some additional information, e.g. the position of the well with the corresponding insert in the Blastema (BL) or Neural tube (NT) library.
• Axologle database contains several different Axolotl transciptome assemblies

Datasets

Next generation sequencing

The data can be found under //biodata/groups/crtd_tanaka/axolotl/Sequencing/Illumina.

• Mature and regenerating spinal cord

  Name	Description	Type	Reads	Location	Author	Remarks
  Mature spinal cord	Mature axolotl spinal cord	Illumina, paired-end, 100bp	65,753,941	SpinalCord/mSC.fastq.gz	Akira Tazaki
  Injured spinal cord	Injured axolotl spinal cord, 6dpi	Illumina, paired-end, 100bp	78,459,950	SpinalCord/6diSC.fastq.gz	Akira Tazaki

• Axolotl limb

  Name	Description	Type	Reads	Location	Author	Remarks
  Mature limb	Mature axolotl forelimb	Illumina, single-end, 76bp	65,934,228	Limb/Mature.fastq.gz	Dunja Knapp
  Amputated limb (36h)	Amputated axolotl forelimb (36hpa)	Illumina, single-end, 76bp	65,250,768	Limb/36h_amp.fastq.gz	Dunja Knapp
  Amputated limb (72h)	Amputated axolotl forelimb (72hpa)	Illumina, single-end, 76bp	62,970,898	Limb/72h_amp.fastq.gz	Dunja Knapp
  Lateral wound (36h)	Injured (lateral wound) axolotl forelimb (36hpw)	Illumina, single-end, 76bp	66,361,470	Limb/36h_lw.fastq.gz	Dunja Knapp
  Lateral wound (72h)	Injured (lateral wound) axolotl forelimb (72hpw)	Illumina, single-end, 76bp	64,133,834	Limb/72h_lw.fastq.gz	Dunja Knapp