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.

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

Antibodies

List of primary antibodies

Name	Supplier	Cat. No	Host	Mono/Poly	Antigen	Contact person	Storage temp	Stock concentration	Conditions for axolotl	Conditions for Xenopus	Conditions for mouse	Conditions for cell culture	Reference
12/101	Developmental Studies Hybridoma Bank		Mouse			Yuka	4°C	46 ug/ml Ig	1:200	1:200
15.3B9 (NOT1)	Developmental Studies Hybridoma Bank	15.3B9 (NOT1)	Mouse	Monoclonal	notochord marker (Chicken)	Yuka	4°C		21 ug/ml IG				http://dshb.biology.uiowa.edu/notochord-marker
20B4	Developmental Studies Hybridoma Bank	20B4	Mouse	Monoclonal	neural crest cell (Chicken)	Yuka	4°C						http://dshb.biology.uiowa.edu/neural-crest-cells
5D3	Developmental Studies Hybridoma Bank	5D3	Mouse	Monoclonal	Cadherin E (Xenopus)	Yuka	4°C		37 ug/ml IG				http://dshb.biology.uiowa.edu/5D3
8C8	Developmental Studies Hybridoma Bank	8C8	Mouse	Monoclonal	integrin beta-1/ CD29 (Xenopus)	Yuka	4°C						http://dshb.biology.uiowa.edu/integrin-beta-1_7
Aggrecan	millipore	AB1031	Rabbit			Lidia				Not work, Dim	'1:500 (Lidia)
Alpha tubulin (DM1a)	MPI		Mouse	Monoclonal	Chicken alpha tubulin	Takuji	-20°C					1:1000 -
b-catenin	Gift from Thomas Kurth	P14L	Rabbit		Xenopus	Yuka_Aliquot from Tomas Kurth							Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos.
Bra		AF2085				Yuka	-20C		0.2ug/ml in PBS
BrdU (Bu20a)	MPI		Mouse	Monoclonal	BrdU - thymidine analog	Takuji	-20°C		1:400 -
BrdU	Antibodies-online	ABIN964591	Rabbit	Polyclonal	BrdU - thymidine analog	Takuji	-20°C		1:600 -				http://www.antibodies-online.com/antibody/964591/anti-Bromodeoxyuridine+BrDU+antibody/
Casp3	Abcam	ab13847	rabbit			Lidia	-20C
col1A2	Developmental Studies Hybridoma Bank	SP1.D8	Mouse	Monoclonal	Sheep	Yuka	-20C	187ug/ml	1:200-	1:200_found in most connective tissue and abundant in bone, cornea, dermis and tendon.
col2A1	Acris	AM00619PU-N	Mouse			Yuka	-20C	0.2mg/ml in /H2O	1:10	1:100-200
Digoxigenin-AP	Roche	11093274910	Sheep	Polyclonal		Common box	4°C						http://www.sigmaaldrich.com/catalog/product/roche/11093274910?lang=de&region=AT&gclid=Cj0KEQjwk-jGBRCbxoPLld_bp-IBEiQAgJaftdEIkv2iCvPxqx7NfQns9MUkipGFBChRXSL1FhsYOxkaApPb8P8HAQ
FITC	Jackson ImmunoResearch	200-002-037	Mouse	Monoclonal	Fluorescein isothiocyanate	Takuji	-20°C		1:400 -				https://www.jacksonimmuno.com/catalog/products/200-002-037
FITC	invitrogen	71-1900	Rabbit	Polyclonal	Fluorescein isothiocyanate	Takuji	-20°C		1:400 -				https://www.thermofisher.com/antibody/product/FITC-Antibody-Polyclonal/71-1900
GFP	Rockland	600-401-215	Rabbit			Common box	-20C			1:2000-4000
GFP	MPI		Goat			Yuka (from Jifeng)	-20C		Yuka (from Jifeng)	1:2000-
GFP	MPI	106-A20-Mix	Mouse			Yuka	-20C	2.96 mg/ml in PBS		1:200, High background
GFP	Abcam	ab13970	Chick			Yuka (from Andrea), Lidia	-20C		Andrea	High background
GFP	Torrey Pines	TP401	Rabbit			Yuka	-20C		1:200 (Axolotl)
GFP	Invitrogen	A11120	Mouse	monoclonal		Common box	-20C	1 mg/ml in PBS
Hoxa11	Tanaka Lab (Kathleen)		Rabbit	Polyclonal	Axolotl	Yuka_Aliquot from Kathleen	-20C	0.95 mg/ml		FF+2%MEMFA.Dent's
Hoxa11	Tanaka Lab (Kathleen)		Mouse	Monoclonal	Axolotl	Yuka_Aliquot from Kathleen	-20C	4.22 mg/ml
Hoxa13	Tanaka Lab (Kathleen)		Rabbit	Polyclonal	Axolotl	Yuka_Aliquot from Kathleen	-20C	0.15 mg/ml	01:50	1:50, FreshFrozen+Unfixed
Hoxa9	Tanaka Lab (Kathleen)		Rabbit	Polyclonal	Axolotl	Yuka_Aliquot from Kathleen	-20C	1.35 mg/ml		FF+Methanol+ph9AR
Laminin	Sigma	L9393	Rabbit			Yuka (from Jifeng)	-20C			1:200, 500_1:25 (paraffin), 1:100 (Slack)
MBP	Genetex	GTX76114	Rat	Monoclonal	Myelin basic protein (Bovine)	Common box	-20C		1:200	1:200
Mef2c	Sigma	HPA005533	Rabbit	Polyclonal	Human MEF2C (Myocyte enhancer factor 2C)	Aliquot from Takuji			1:50 (Prayag), 1:300 (Takuji)	1:5000,10000 (High background on all type of tissue)
Meis 1/2/3	Millipore	05-779	Mouse IgG1	Monoclonal	mouse Meis 1 (aa 60-390)	Lidia	-20C (30% glycerol)		1:200 (Takuji?- frozen tissue secitons)		1:200 (Lidia?)		http://www.merckmillipore.com/AT/de/product/Anti-Meis-1%2F2%2F3-Antibody%2C-clone-9.2.7,MM_NF-05-779?ReferrerURL=https%3A%2F%2Fwww.google.at%2F&bd=1
MHC	Developmental Studies Hybridoma Bank	A4.1025	Mouse			Yuka	4°C		1:200	1:200
Osterix	abcam	ab22552	rabbbit			Lidia
Pax7	MPI		Mouse			Yuka	-20C		1:100, 200, 400
PCNA	Santa Cruz	sc-56 AF64	Alexa Fluor -647			Lidia				1:500
PDGFRa (16A1) - Biotin	Invitrogen	A15732	Mouse	Monoclonal	Human CD104a	Yuka (from Josh)	4°C	1 mg/ml					https://www.thermofisher.com/antibody/product/PDGFRA-Antibody-clone-16A1-Monoclonal/A15732
Perilipin	R and D	P1873	rabbit			Lidia
PHH3	millipore	06-570	rabbit	Polyclonal	Linear peptide corresponding to human Histone H3 at Ser10	Takuji	4°C	1 mg/ml	1:400 -				https://www.merckmillipore.com/DE/de/product/Anti-phospho-Histone-H3-%28Ser10%29-Antibody%2C-Mitosis-Marker,MM_NF-06-570?ReferrerURL=https%3A%2F%2Fwww.emdmillipore.com%2FCA%2Fen%2Fproduct%2FAnti-phospho-Histone-H3-%2528Ser10%2529-Antibody%252C-Mitosis-Marker%2CMM_NF-06-570
PPARgamma	Cell signalling	81B8	rabbit			Lidia
Prrx1	Tanaka Lab (Prayag)		Rabbit	Polyclonal	Axolotl N-terminus (1-101 aa)	Lidia, Yuka_Aliquot from Prayag	-20C		1:200	1:200
RFP	Rockland	600-401-379	Rabbit	Polyclonal		Common box	-20C		1:300-1:1000 for Axolotl	1:1000 or 1:2000			http://www.rockland-inc.com/Product.aspx?id=34801
Rhodamine	Molecular Probes	A-6397	Rabbit	Polyclonal	Tetramethylrhodamine	Takuji	-20°C		1:400 -
Scleraxis	Santa Cruz	D-14, sc-87425	Goat	Polyclonal Goat IgG		Yuka	4°C
Shh (H-160)	Santa Cruz	SC-9024	Rabbit	Polyclonal		Yuka	4°C		200 ug/ml
Sox9	Chemicon	Ab5535	Rabbit	Polyclonal	Synthetic peptide from human Sox9, aa486-509 (extreme C-Terminus)	Yuka_Aliquot	-20C		1:500-1:1000	1:500			http://www.merckmillipore.com/DE/en/product/Anti-Sox9-Antibody,MM_NF-AB5535
Sox9	R&D	AF3075	Goat	Polyclonal	E. coli-derived recombinant human SOX9 (Met1-Lys151)	Lidia, Yuka	-20C		1:100-1:200	1:100			https://www.rndsystems.com/products/human-sox9-antibody_af3075
Zo-1	Invitrogen	33-9100	Mouse			Yuka (from Akira)	-20C
GADD45B	SIGMA	SAB2108614-100	Rabbit	Polyclonal	QIHFT LIQSF CCDND INIVR VSGMQ RLAQL LGEPA ETQGT TEARD LHCLL	Marco	-20C	0.5-1 mg/ml
GADD45G	SIGMA	HPA023606	Rabbit	Polyclonal	MTLEE VRGQD TVPES TARMQ GAGKA LHELL LSAQR QGCLT AGVYE SAKVL NVDPD NVTFC VLAAG EEDEG DIALQ IHFTL IQAFC CENDI DIVRV GDVQR LAAIV G	Marco	-20C	0.05mg/ml
TDG	SIGMA	HPA052263	Rabbit	Polyclonal	WKCLF MSGLS EVQLN HMDDH TLPGK YGIGF TNMVE RTTPG SKDLS SKEFR EGGRI LVQKL QKYQP RIAVF NGKCI YEIFS KEVFG VKVKN LEFG		-20C	0.1mg/ml
Tet3	Diagenode	C15410311	Rabbit	Polyclonal		Marco	-20C	1 ug/uL
Tet2	Diagenode	C15410255	Rabbit	Polyclonal		Marco	-20C	1 ug/uL
5-mC	Diagenode	C15200081-100	Mouse	Monoclonal		Marco	-20C	1.36 ug/ uL
5-hmC	Diagenode	C15410205-20	Rabbit	Polyclonal		Marco	-20C	3.5 ug/uL
IgG (Rabbit)	Diagenode	C15410206	Rabbit	Polyclonal		Marco	-20C	1 ug/uL
IgG (Rabbit)	CST	2729S	Rabbit	Polyclonal		Akane	-20C	1-5ug/uL					https://media.cellsignal.com/pdf/2729.pdf#_ga=2.22008443.1736501072.1510317731-1668154722.1509367929
anti-H3 (Rabbit)	Abcam	ab1791	Rabbit	Polyclonal		Akane	-20C	1 ug/uL	1:5000(WB), 1:1000 (CHiP), 1:1000 (Tissue IF)				Synthetic peptide corresponding to Human Histone H3 aa 100 to the C-terminus conjugated to Keyhole Limpet Haemocyanin (KLH).http://www.abcam.co.jp/histone-h3-antibody-nuclear-loading-control-and-chip-grade-ab1791.html
anti-H3K4me3 (Rabbit)	Abcam	ab8580	Rabbit	Polyclonal		Akane	-20C	1 ug/uL	1:1000 (WB), 1:250 (CHiP), 1:500 (Tissue IF)				Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.(http://www.abcam.co.jp/histone-h3-tri-methyl-k4-antibody-chip-grade-ab8580.html)
anti-anti-H3K4me1 (Rabbit)	Abcam	ab8895	Rabbit	Polyclonal		Akane	-20C	0,2 ug/uL	1:500 (WB), 1:250 (CHiP), 1:500 (Tissue IF)				Synthetic peptide within Human Histone H3 aa 1-100 (mono methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH). http://www.abcam.co.jp/histone-h3-mono-methyl-k4-antibody-chip-grade-ab8895.html
anti-H3K9me3 (Rabbit)	Abcam	ab8898	Rabbit	Polyclonal		Akane	-20C	1 ug/uL	1:1000 (WB), 1:500 (Tissue IF)				Synthetic peptide within Human Histone H3 aa 1-100 (N terminal) (tri methyl K9) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary. (Peptide available as ab1773) http://www.abcam.co.jp/histone-h3-tri-methyl-k9-antibody-chip-grade-ab8898.html%7C%7C
anti-H3K27ac (Rabbit)	Abcam	ab4729	Rabbit	Polyclonal		Akane	-20C	1 ug/uL	1:1000 (WB), 1:500 (Tissue IF)				Synthetic peptide corresponding to Human Histone H3 aa 1-100 (acetyl K27) conjugated to Keyhole Limpet Haemocyanin (KLH). http://www.abcam.co.jp/histone-h3-acetyl-k27-antibody-chip-grade-ab4729.html
anti-H3K27me3 (Rabbit)	millipore	07-449	Rabbit	Polyclonal		Akane	-20C	1 ug/uL	1:1000 (WB), 1:500 (Tissue IF)				KLH-conjugated, synthetic 2X-branched peptide containing the sequence …AR(me3K)SAP… in which me3K corresponds to trimethyl-lysine at residue 27 of human histone H3. http://www.merckmillipore.com/AT/de/product/Anti-trimethyl-Histone-H3-Lys27-Antibody,MM_NF-07-449?ReferrerURL=https%3A%2F%2Fwww.google.at%2F&bd=1
anti-Ezh2 mAB (mouse)	CST	CST3147	mouse	mono clonal		Akane	-20C	1 ug/uL	WB, IF, ChIP DIDNT WORK				https://www.cellsignal.com/products/primary-antibodies/ezh2-ac22-mouse-mab/3147
Pol II antibody (mouse)	CST	C15100055-100	mouse	mono clonal		Akane	-20C	1 ug/uL					mouse against the B1 subunit of RNA polymerase II (polymerase (RNA) II (DNA directed) polypeptide A) of wheat germ. Interacts with the highly conserved C-terminal domain of the protein containing the YSPTSPS repeat. https://www.diagenode.com/files/products/antibodies/Datasheet_PoII_C15100055.pdf
IgG (mouse)	CST		Rabit	Mono clonal		Akane	-20C	1 ug/uL	WB, IF, ChIP DIDNT WORK
CTCF (D31H2) XP®	CST	CST-3418S	Rabbit	Mono clonal		Akane	-20C	1 ug/uL	WB, IF, ChIP DIDNT WORK

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

Antibodies

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

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.

Literature

• Theses
• Publications

Databases

• Axolotl transcriptome website contains the Axolotl transcriptome assemblies
• Axolotl genome website contains the Axolotl genome 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
• Axologle database contains expression profiling data of Axolotl limb regeneration
• 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.