- Surgical drill. Tungsten carbide drill (KOMPH141010 for
stereotaxic surgery and KOMPH141014 for intracerebroven-
tricular (ICV) surgery, Komet Dental). - Cyanoacrylate adhesive.
- Gavage needle for rat (Fine Science Tools, Item No. 18061-
10.) - Hamilton syringe for ICV (Hamilton, model #7001N
22 gauge). - Hamilton syringe for rat stereotaxic administration (Hamilton,
model #7001N 25 gauge). - Hamilton syringe for mouse stereotaxic administration
(Hamilton, model #7001KH 32 gauge). - Osmotic minipumps (Alzet, models 2001, 1007D, 1004D).
- Stereotaxic frame.
- Silk suture (rat/mouse) 2-0/4-0.
2.2 Dissection
and Protein Extraction
- PBS (phosphate buffer saline): 138 mM NaCl, 2.7 mM KCl,
1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 ·7H 2 O, pH 7.4. - Precision scissors.
- Surgical blade stainless steel n15.
- Razor blades.
- 1 mm coronal acrylic brain matrix for rat or mouse.
- Lysis buffer with protease inhibitor (BLYSpi): 50 mM
Tris–HCl, pH 7.5, 10 mM EGTA, 10 mM EDTA, 1% (v/v)
M Triton X-100, 1 mM sodium orthovanadate, 50 mM
sodium fluoride, 10 mM sodium pyrophosphate, 0.25 M
sucrose, protease inhibitor cocktail. Store at 4C. Weight
6.057 g Tris–HCl, 0.38 g EGTA, 0.37 g EDTA, 0.184 g
sodium orthovanadate, 2.099 g sodium fluoride, 2.23 g
sodium pyrophosphate, 85.5 g sucrose. Add 10 mL of Triton
X-100. Make up to 1 L with distilled water. Mix and adjust
pH 7.5 with HCl (seeNote 1). Use one tablet of protease
inhibitor in 50 mL of lysis buffer (seeNote 2). - Stainless steel grinding beads and bead mill homogenizer.
- 1 mg/mL bovine serum albumin (BSA) in distilled water.
- Bradford solution (protein assay dye reagent concentrate).
Dilute in distilled water 1:4. Prepare enough for 250μL for
each sample and standard in duplicate. Keep protected from
light. Incubate 10 min at 37C before use. - 96-well plate: clear plates for colorimetric assay.
- Microplater reader. Thermo Fisher multiskan go.
Methods for Brain AMPK 435