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

Maintainance

In this section you will find some general information on how to maintain the animals.

Mating

In this section you will find the information on how to mate axolotls.

Feeding

This section will give you an overview of how to feed the axolotls.

Axolotl in Vitro Fertilization

This section will give you an overview of how to In vitro fertilize the axolotls.

Interesting observations

In the following section you can find some interesting observations concerning the axolotl, its behavior, caveats of the transgenics and many more.

Genotyping (galaxy)

This section section will give you an overview of how to genotype the axolotls.

Lab duties

Chemicals

• Antibiotics
• Benzocaine
• Fibronectin
• Gelatine
• Liquid nitrogen
• Primary antibodies
• Secondary antibodies
• Serum heat inactivation

Hardware

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

General

• Ordering common stuff
• Heat-shock competent cells

Protocols

Cell culture

• Adhesive cell culture
• Baculovirus titration
• Cardiomyocyte preparation
• Electroporation of neural epithelia cells (axolotl spinal cord)
• Myoblasts electroporation
• Neurosphere culture
• Thawing 293FT cells
• G418 medium preparation
• Fibronectin preparation
• Gelatin preparation
• Insulin preparation
• Insulin preparation
• Gelatin Embedding

A1 cells

• Thawing frozen A1 cells
• Freezing of A1 cells
• A1 cell passaging
• A1 cell passaging for myotube preparation
• Modified myotube prep
• A1 cell transfection with FuGene
• Electroporation of A1 cells
• BrdU labelling of A1 cells in 96-well plate
• BrdU and myosin staining
• Preparation of Low Serum for A1 cells
• Preparation of High Serum for A1 cells
• Preparation of Freezing media for A1 cells

Blastema cells

• Blastema cells
• Electroporation of blastema cells

C2C12 cells

• Thawing frozen C2C12 cells
• Freezing of C2C12 cells
• C2C12 cell passaging
• Myotube preparation of C2C12 cells
• C2C12 cell transfection with FuGene
• Cloning of C2C12 cells
• Electroporation of C2C12 cells
• Preparation of Low Serum for C2C12 cells
• Preparation of High Serum for C2C12 cells
• Preparation of Freezing media for C2C12 cells

Embryonic stem cells

• mESC culture/Cyst formation

Hybridoma cells

• Thawing (XB10) hybridoma cells
• Hybridoma cells in SFX media
• Hybridoma cell clones

Histology

• Alcian Blue Staining
• Double Immunostaining via antigen retrieval (Sherry)
• Tyramide signal amplification (TSA) staining on axolotl tissue

In situ Hybridization

• ISH on axolotl tissue sections (Anja)
• ISH on axolotl tissue sections (Akira)
• Whole mount ISH on axolotl tissue

Antibody

Monoclonal Antibodies:

• Purifying IgG1 from Hybridoma Supernatant
• Labelling myosin antibody
• Labelling Pax6 with Dig-NHS
• Anti-BrDU Antibody labeling

Polyclonal Antibodies:

• Address for making polyclonal antibodies
• Letter to Froppier (French)
• labeled AB staining
• Antibody staining
• Antibody purification
• Antibody purification Sox2

ELISA:

• ELISA

Molecular biology

• Metamorphosis protocol
• Plasmid digestion
• RNA Extraction (axolotl tissue)
• RNA formaldehyde gels
• Amputation and RNA Isolation
• Linker Insertion
• gRNA Production
• Cocktail Protocol for Pax7

Lab business

Ordering database

Use the following ordering database to place your orders.

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 and dissecting microscope
  • Room 0.232
    • Nepa Gene
    • ECM 830
    • Olympus dissecting microscope
  • Lab
    • Olympus (next to the axolotls)
    • Olympus (next to the Microtome)
• Confocal microscopes
  • Leica LSM
• Cryostats
  • Cryostat 1
  • Cryostat 2
• Microtome
  • Microtome
• Paraffin embedding station
  • Paraffin embedding station

Genetics

Transgenic lines

• Axolotl (A.mexicanum)
• Frog (X.laevis)

Markers

Marker	Full name	Target	Remarks	Link
DAPI/Hoechst	4',6-diamidino-2-phenylindole	Nuclei	Incorporates into the DNA	DAPI, Hoechst
PCNA	Proliferating-Cell-Nuclear-Antigen	Dividing cells		PCNA
SOX2	SRY (sex determining region Y)-box 2	Preferentially neuronal lineage	Progenitor marker for both spinal cord and brain	SOX2
PAX6	Paired box protein Pax-6	Brain/spinal cord	aka aniridia type II protein (AN2) or oculorhombin	PAX6
PAX7	Paired box protein Pax-7	Satellite cells/spinal cord/brain		PAX7
MHC	Myosin heavy chain	Muscle fibers		MHC
MYF5	Myogenic factor 5	Myoblasts	Regulates muscle differentiation, labels cycling myoblasts	MYF5
TUBB3	ßIII-tubulin	Axons		TUBB3
MBP	Myelin basic protein	Schwann cells
PRRX1	Paired mesoderm homeobox protein 1	Connective tissue progenitors in Limb bud and Limb Blastema		PRRX1
SMA	Smooth muscle actin	Blood vessels/endothelial cells
GFAP	Glial fibrillary acidic protein	Glia		GFAP
NeuN	Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3	Differentiated nerve		NeuN
LEU7/HNK1/B3GAT1/CD57	Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1	Schwann cells		B3GAT1
XBRA	Xbrachyury	Mesoderm during gastrula stage		Brachyury
MSX1	Muscle segment homeobox, Msh homeobox 1	Undifferentiated cells	Transcriptional repressor during embryogenesis	MSX1
Trypan blue		Dead tissues or cells	Vital stain	Trypan blue
MEF2C	Myocyte-specific enhancer factor 2C	Differentiated muscle cells	aka MADS box transcription enhancer factor 2, polypeptide C	MEF2C
PH3	Phospho-Histone H3	Mitotic cells
BrdU	Bromodeoxyuridine	Cells in S-phase		BrdU
CASP3	Caspase 3	Dying cells		CASP3
GDF5	Growth/differentiation factor 5	Joints	Joint development marker	GDF5
Survivin		Cleavage furrow	aka baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5	Survivin
MAP2	Microtubule-associated protein 2	Neurons		MAP2
SOX9	SRY (sex determining region Y)-box 2	Cartillage progenitors		SOX9
MBP	Myelin basic protein	Myelin sheath		MBP

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.

Fileservers

Location	Volume size	Description	Access	Location (console only)
//biodata/groups/crtd_tanaka	16 TB	General purpose storage space	Entire Tanaka lab	biocluster: /group/crtd_tanaka
//biodata/groups/tanaka-ests	247 GB	EST sequences	Entire Tanaka lab	biocluster: /group/tanaka-ests
//biodata/groups/axolotl-transcriptome	500 GB	Axolotl transcriptome data	Elly, Sergej	biocluster: /projects/axolotl-transcriptome
//biodata/groups/tanaka-saori	878 GB	Special directory for Saori's data	Elly, Saori, Sergej	biocluster: /group/tanaka-saori

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NOTE: The path is different, when accessing the file server from the console.

Literature

• Theses
• Publications

Databases

• Axolotl transcriptome website contains the Axolotl transcriptome assemblies
• Newt transcriptome website contains the Newt transcriptome assembly
• Red spotted newt transcriptome website contains the red spotted newt transcriptome assembly
• 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.
• Short insert cDNA library
• CURRENTLY UNAVAILABLE! Axologle database contains expression profiling data of Axolotl limb regeneration
• CURRENTLY UNAVAILABLE! Axolotl BLAST database contains several different BLAST-able Axolotl transciptome assemblies

Datasets

• Sequences
  • Illumina
  • Sanger
  • Roche/454
• Microarrays

Interesting links

• Visible in the invisible: a movie introducing a new image processing ana analysis technique developed at the MIT.

Lab life

• Lab photos
• Further pictures can be found in /biodata/groups/crtd_tanaka/LabFun/
• The recepies for Heino's farewell present can be found under /biodata/groups/crtd_tanaka/LabFun/Heino's farewell present/
• The video of the visit of the EU commissioner can be found under /biodata/groups/crtd_tanaka/LabFun/