Dulbecco's Modified Eagle Medium, high glucose (DMEM:HG) (ThermoFisher Scientific, Cat no. 11995065)
Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat no. 14190144)
Trypsin-EDTA (ThermoFisher Scientific, Cat no. MT25053CI)
Trypan Blue (ThermoFisher Scientific, Cat no.T10282)
Fetal Bovine Serum (VWR 89510-188)
100X Non essential amino acids (Fisher Scientific, Cat no. 11-140-050)
Milli Q water
16% paraformaldehyde (Electron Microscopy Sciences, EMS, Cat no. 15700)
25% glutaraldehyde (Electron Microscopy Sciences, Cat no. 16019)
0.4 M sodium cacodylate buffer, pH 7.2 (Electron Microscopy Sciences, Cat no. 11654)
4% Osmium Tetroxide (Electron Microscopy Sciences, Cat no. 19150)
Potassium Ferrocyanide (same as potassium hexacyanoferrate (II) trihydrate, Sigma, Cat no. 60279)
Thiocarbohydrazide (TCH, Electron Microscopy Sciences, Cat no. 21900)
L-aspartic acid (Sigma-Aldrich, Cat no. A9256)
Lead nitrate (Ted Pella Inc., Cat no. 19321)
Ethanol, 200 proof (Electron Microscopy Sciences, Cat no. 15056)
Durcupan ACM Epoxy (Electron Microscopy Science, Cat no. 14040)
EMbed 812 Embedding Kit (Electron Microscopy Science, Cat no. 14121)
Silver conductive epoxy (Chemtronics, Cat no. CW2400 Epoxy)
Biosafety cabinet
CO2 incubator (Heracell CO2 Incubators, ThermoFisher Scientific)
Inverted light microscope to check confluence of cells (Carl Zeiss Microscopy LLC)
Countess II Cell Counter (ThermoFisher Scientific)
Invitrogen Countess Cell Counting Chamber Slides (ThermoFisher Scientific, Cat no. C10312)
Ibitreat, 35 mm tissue culture treated dish (Ibidi, Cat no. 81156)
35 mm tissue culture dish; No. 1.5 Gridded Coverslip; 14 mm Glass Diameter (MatTek Corporation, Cat no. P35G-1.5-14-CGRD)
Cell scraper (VWR 10062-904)
Wood Applicator Sticks (Solon, Cat no. FBHOO360)
Embedding mold (PELCO, Cat no. 10535)
Pipette and pipette tips
Chemical hoods
Razor (EMS Cat# 72000-WA)
Formar Support Copper Slot Grid (Electron Microscopy Science, Cat no. FF2010)
Complete DMEM: HG media containing 10% fetal bovine serum and 1X non essential amino acids.
Complete DMEM: HG media containing 3% fetal bovine serum and 1X non essential amino acids.
Fixation solution: 2.5% Glutaraldehyde, 2% paraformaldehyde in 0.1M Sodium Cacodylate Buffer (pH 7.2-7.4). The osmolarity of the original Karnovsky mixture (4% PFA and 5% glutaraldehyde) is too high (2010 mOsmol), therefore, half strength (2% PFA and 2.5% glutaraldehyde) of the aldehyde mixture is more popular today.
TCH solution: 0.1g TCH to 10 mL ddH2O and place in a 60 °C oven for 1 h, agitate by swirling gently every 10 mins to facilitate dissolving. Filter solution through a 0.22 μm filter right before use.
2% Osmium in ddH2O
1% Uranyl acetate in ddH2O
1.5% potassium ferrocyanide in ddH2O
Aspartic acid stock solution: dissolve 0.998 g of L-aspartic acid in 250 mL ddH2O water. Note: aspartic acid dissolves more readily at pH 3.8. The solution is stable for 1-2 months at 4 °C.
Walton’s lead aspartate solution[1]: dissolve 0.066g lead nitrate in 10 mL of the aspartic acid stock solution. Adjust the pH of the solution to 5.5 with 1M KOH. Durcupan ACM: Epoxy resin/ A (11.4 g); 964 hardener/ B (10 g); 964 accelerator/ C (0.3 g); Dibutyl phthalate / D (0.05-0.1 g) for en face samples
Hard Epon: EMbed[2] 812 (5 mL); DDSA (2.25 mL); NMA (3 mL); BDMA (0.3 mL) for cell pellet samples.