RABINOVITCH PROTOCOL
MANUAL
U:/PUBLIC/Lab Manual/Rabinovitch Manual 2005
Preparing Chemical Waste for Pickup and
Disposal
Problems
with Cooling, Heating, the Building or Scientic Instruments
Chilled Water Failure in Flow Cytometry Room
General Heating, Cooling, Fumehood, or
Building Problems:
SECTION 2
Record Keeping, Animal Use AND
Computer Use
Tissue
Culture Media Preparation
Calculation
of Population doulbing
Esophageal
Squamous Cell Media
Starting
Primary Cultures from Barrett’s Esophagus Biopsies
Set
up of Esophageal Squamous cultures
Soft
Agar Assay for Colony Formation..
Lymphocyte
Preparation from Whole Blood
Preparation
of Mouse Embryo Fibroblast Cell Strains
Fixation
and embedding cultured cells in agarose for paraffin
Section 4 Maintenance and calibration of
Incubators
Testing
CO2 levels in the incubators
Section 5 Flow protocols For Apoptosis and
Proliferative Survival
Hoechst
33342/SYTO 11 Protocol for Apoptosis
Hoechst
33342/SYTO 11/CMXRosamine Protocol for Apoptosis
Hoechst
33342/SYTO 11/PI Protocol for Apoptosis
Hoechst
33342/SYTO 11/PI/CEN Protocol for
Apoptosis
Processing data by the Hoechst 33342/SYTO
11/PI/CEN Protocol for Apoptosis
Hoechst
33342/MTG/CMXRosamine Protocol for Apoptosis
Proliferative
Survival Protocol
Processing data for the Proliferative
Survival Protocol
Section 6 Protocols for FISH and Antibody
Stains
Deparaffinization
of tissues on slides
Assay
for Detection of Cyclin B by Flow Cytometry (small cell number)
Double
Staining with Cyclin B and Pericentrin (sequential staining)
Epi
shake off procedure to isolate G1 cells for FISH and G2 cells for anaphase
bridge scoring
Epi
shake off: isolation of epithelial cells
Tissue
Digestion and CK Staining
Slide
Fixation for CK-sorted cells dropped from Flow Cytometer
Fixation
of Sorted Cells for FISH
Digoxigenin
Labeling of BAC DNA for FISH
Preparation,
dropping, and fixing of lymphocyte metaphase slides
Staining
of Tissue OR CELLS WITH CATALASE (can be used for most antibody stains)
RC
DC Assay Protein Quantification..
OXYBLOT
PROTEIN OXIDATION DETECTION
Section 7 Protocols to evaluate telomere
length
Preparation
of Tissue for Telomere Probe
Light fixation of fresh or frozen tissue
Hybridization
PNA and Amadate Probes
Confocal
Microscope set up for photos
Protocol
For Image Analysis Using Optimas 6.51
EXCEL
MACRO INSTRUCTIONS FOR THE ANALYSIS OF OPTIMAS DATA
TERMINAL
RESTRICTION FRAGMENT (TRF) PROTOCOL
Section 8 BAC cloning protocols
Plasmid
Mini-Prep: Preparation of BAC DNA for
analytical purposes.
Section 9
Flow Protocols for Cell Cycle, Proliferation, Viability and Coast Sea
Food.
Hoescht/Fluorescein
Diacetate (FDA)/Propidium Iodide (PI)
Section 10 Protocols for Arrays and RNA preps
for Arrays
Basic
RNA extraction detailed protocol
RNA
Extraction from Pancreatic Tissue (LiCl/RNeasy combination method)
General
Instructions for Bioanalyzer RNA
Preparation of cDNA from total RNA (Reverse Transcription, RT)
ENZO
RNA prep for generation of cRNA..
AFFYmetrix
RNA prep for generation of cRNA using IVT kit
Section 11
Macros, SAM, GeneTraffic hints
Entropy Macro “SID.xlm” (general purpose)
Entropy Macro “SID.xlm” (Telomere arm gain_loss, centromere gain_loss)