Contents |
On PD 35 EUTHANIZE ANIMAL VIA CO2 AND DECAPITATION
DISSECT THE SCULL AND REMOVE THE BRAIN
TRIM UNWANTED BRAIN REGIONS USING STERIOTAXIC BLOCK
IMMERSE BRAIN IN 2-METHYLBUTANE AT -20° to -25°C FOR 30 SECONDS
GENTLY WRAP BRAIN IN CELOPHANE THEN ALUMINUM FOIL AND LABLE
PLACE IN DRY ICE COOLER AND TRANSFER TO -80°C FREEZER
TRANSFER BRAIN TO CRYOSTAT SET AT -20°C AND ALLOW TIME FOR BRAIN TO ACCLIMATE TO THIS TEMPERATURE
PLACE DISPOSIBLE BLADES INTO CRYOSTAT AND ALLOW THEM TO ACCLIMATE TO THIS TEMPERATURE
LABLE POSITIVELY CHARGED SLIDES AND COOL THEM TO 4°C
MOUNT BRAIN ON CHUCK USING OCT COMPOUND USING ENOUGH TO COAT ENTIRE BRAIN
INSERT BLADE INTO BLADE-HOLDER – BE SURE THAT IT IS NOT CLAMPED TOO TIGHT, OR THIS WILL CAUSE THE BLADE TO BOW
DESTATIC BRUSHES, BLADE, AND ANTI-ROLL PLATE CONTINUOUSLY
SET TRIM AND SECTION THICKNESS TO DESIRED MEASURE CUT FOR T AND NS
AFTER CUTTING, PLACE SLIDES IN OPEN SLIDE BOX
PUT DESSICANT IN TUPPERWARE WITH KIM WIPE ABOVE DESSICANT
PLACE SLIDE BOX ONTOP OF KIM WIPES AND SEAL THE TUPPERWARE CONTAINER WITH PARAFILM TO KEEP AIR TIGHT
KEEP AT 4°C FOR 2 HOURS
TRANSFER TO -20°C FOR OVERNIGHT STOREAGE
SECTIONS SHOULD NOT BE STORED FOR MORE THAN 30 DAYS
Twenty-micron coronal sections, cut at -20°C and thaw-mounted onto cold, positively charged, glass slides. For each radioligand, 3 sections are taken at 220 μm intervals throughout the hippocampus. These sections will be taken at -3.80 mm bregma to -2.80 mm bregma (where the ventral hippocampus is no longer present). This will yield ~9 sections per brain. One section will be used for specific binding, one for nonspecific binding, and one for nissel staining.
REMOVE SLIDES FROM FREEZER AND LEAVE AT ROOM TEMPERATURE FOR AT LEAST 30 MINUTES. MAKE PREINCUBATION, INCUBATION, AND WASH OUT SOLUTIONS ACCORDING TO PROTOCOL.
EXAMPLE M1: Buffer – do all calculations first. Determine number of slides, and how many slides of each S and NS will fit into each bath apparatus 20-Slide Baths require 200 mL buffer each
Step 1 List all molar ratio’s and molecular weights of compounds according to bottles For 1 L make HEPES buffer in the following quantities: 0.1 M NaCl – 58.44 g/mol 2.0 mM KH2PO4 – 136.0 g/mol 1.2 mM MgCl2 – 203.31 g/mol 2.5 mM CaCl2 – 147.012 g/mol 5.0 mM HEPES – 238.31 g/mol 5.5 mM Glucose – 180.16 g/mol
Step 2 List desired quantities and convert to grams: KH2PO4 2.0 mmol / 1000 mmol * 1 mol * 1 L / 1mol * 136 g = 0.272 g The 1 L above is replaced by whatever the desired total volume is Do this step for all compounds
Mix the calculated quantities into a glass beaker with stir-bar and appropriate amount of dH2O After all solutions are dissolved, use the pH meter to test the pH of the buffer (for M1 it is 7.4). If pH is to high, add concentrated acid, if too low, add concentrated base. Quantities for M1 Pre-incubation: Bath format ~200 mL for the 20-slide bath Make separate baths for the S and NS by separating unliganded buffer before adding any NS ligand (atropine). S = tritium NS = atropine + tritium Incubation: Pipette format 1 mL solution for each slide For 64 total slides, 32-S 32-NS This means ~35 mL buffer with tritium and ~35 mL buffer with atropine + tritium Wash: Same quantities as pre-incubation multiplied X2 2 times 2 min bathes 1 dip in dH2O at 4°C
Take the buffer set aside for incubation (1 mL/slide) and bring it to the ventilation hood for adding the NS-radioligand. This should be done using aluminum foil wrapped vials, in order to shield the ligand from the light (it may be light sensitive). After tritium ligand is added, separate into two containers, 1 for the S and one for the NS. Then add the NS to one of the two. Clearly label. For M1 the NS is atropine at 1 μL and the S is pirenzepine at 10 nM
Put a moist towel inside the incubation box, otherwise pipetted ligand will evaporate Level incubation box, and prepare aluminum foil covering Wipe all sides of slide so liquid will not spill off when pipetted Pipette 1 mL of S or NS to the appropriate slides, be sure slides are not touching before pipetting (use PAP pen if necessary) Cover box with foil Wait appropriate time After incubation period, drain excess ligand off slide and into a marked container Do baths and dips at the appropriate temperature and for the appropriate amount of time Set slides in an upright holder and allow them to dry overnight
LAY FILM ON TOP OF SLIDE, EMULSION SIDE FACING TISSUE SANDWICH SLIDE AND FILM WITH BLANK SLIDE CLAMP BOTH ENDS WITH PAPER CLAMP PUT IN BLACK BOX TAPE AND COVER IN ALUMINUM When it comes time to develop, develop one at a time to determine optimum time to develop the full sheets of film
Store cassettes at room temperature
Develop film 1:1 D-19 4 minutes 2% Acetic Acid 30 seconds Rapid Fix 2 minutes ddH2O 20 minutes Hang film until dry
Radioligand binding CHT:
[3HlHemicholinium ([3H]HC) (147.5 Ci/mMol) is used to label presynaptic high affinity choline uptake sites (HACU) using a modification of the method of Bekenstein and Wooten. In the present study, nonspecific binding is decreased by inclusion of 0.005% polyethylineamine (PEI) in the preincubation buffer and increasing the wash-out time from 30 s to 2 min. These modifications increased specific binding to approximately 70% of total binding.
1 slide = 1ml solution
[3H]Hemicholinium 10 nM
Pre-incubation buffer 50 mM gylcylglicine 200 mM NaCl 0.005% polyethylenimine (PEI) pH 7.8 2 x 5 min at 22 C
Incubation buffer 50 mM gylcylglicine 200 mM NaCl pH 7.8 30 min at 22 C
Washout 50 mM gylcylglicine 200 mM NaCl 0.005% polyethylenimine pH 7.8 2 min at 4 C 3 dips in dH2O
Radioligand binding Muscarinic M1 Receptors:
Radioligand [3H]pirenzepine 10nM a,b
Non-specific label Atropine 1μM
Pre-incubation Krebs-Hepes pH 7.4 10 min at 22°C
Incubation Same buffer 60 min at 22°C
Wash Same buffer 2 x 2 min 1 dip in ddH2O at 4°C
Radioligand binding Muscarinic M2 Receptors:
Radioligand [3H]AF-DX 384 5 nm a,b
Non-specific label Atropine 1μM
Pre-incubation Krebs-Hepes pH 7.4 15 min at 22°C
Incubation Same buffer 120 min at 22°C
Wash Same buffer 2 x 5 min 1 dip in ddH2O at 4°C
Krebs-Hepes Buffer Solution M NaCl 2.0 mM KCL 1.2 mM KH2PO4 1.2 mM MgCl2 2.5 mM CaCl2 5.0 mM HEPES 5.5 mM glucose
Quantitative autoradiography.
Autoradiographic images are quantitated using a video-based computerized image analysis system. A calibration curve of optical density versus radioactivity [femtomoles per milligram (fmol/mg) of tissue wet weight] is generated using the plastic standards. Optical densities in discrete brain regions are measured and the corresponding radioligand binding values determined by interpolation from the standard curve. Radioligand binding sites are anatomically localized by comparing the autoradiographic image and the corresponding nissl-stained tissue section to the rat brain atlas. Data is collected through multiple readings that cover the extent of each brain structure contained within one predetermined coronal level. The particular coronal level chosen for a given structure was defined by anatomical limits, which should not vary between brains or radioligands.