TABLE 2.
Summary protocol, axonal radius and number of mitochondria
| Figure₁ | Species | Genotype/Condition | Tissue | Protocol₃ | Perf/Fix | Postfix [duration] | N | Correlation axonal radius versus mitosize | Mitochondria numbers per axon | Correlation g‐ratio versus mitosize | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| r 2 | p value | Median | Min | Max | r 2 | p value | ||||||||
| 1C₁ | Mouse | Naive | Corpus callosum | ll | 2.5%GA | 82 | 0.2772 | <0.0001 | 1 | 1 | 5 | 0.2259 | <0.0001 | |
| 1C₁ | Mouse | Naive | Corpus callosum | mm | 2.%PFA/2.5%GA | 2.%PFA/2.5%GA [overnight] | 22 | 0.0645 | 0.2539 | 1 | 1 | 4 | 0.2745 | 0.0123 |
| 1D | Rat | Naive | Corpus callosum | aa | 1%PFA/2.5%GA | 2% OsO4 [2 hr] | 61 | 0.2957 | <0.0001 | 1 | 1 | 2 | 0.3978 | <0.0001 |
| 1E₁ | Macaque | Naive | Spinal cord | cc | 2%Form/2.5%GA | 29 | 0.0341₂ | 0.1258₂ | 1 | 1 | 1 | 0.4428₂ | <0.0001₂ | |
| 1E₁ | Macaque | Naive | Spinal cord | bb | 2%PFA/2%GA | 43 | 1 | 1 | 1 | |||||
| 1F₁ | Human | Naive | Prefrontal cortex | dd | 4%PFA/2.5%GA | 4%PFA/2.5%GA [1 week] | 7 | 0.0014₂ | 0.8346₂ | 1 | 1 | 1 | 0.1304₂ | 0.0459₂ |
| 1F₁ | Human | Naive | Prefrontal cortex | dd | 4%PFA/2.5%GA | 4%PFA/2.5%GA [1 week] | 25 | 1 | 1 | 1 | ||||
| 2A | Rat | LPC 10 days | Corpus callosum | aa | 1%PFA/2.5%GA | 2% OsO4 [2 hr] | 39 | 0.1863 | 0.3164 | 2 | 1 | 4 | 0.0176 | 0.4214 |
| 2B | Rat | LPC 24 dpi | Spinal cord | ee | 2%Form/2.5%GA | 4%Form [4–10 days] | 55 | 0.0326 | 0.2098 | 3 | 1 | 5 | 0.1994 | 0.0012 |
| 2C₁ | Mouse | Cuprizone 5 weeks | Corpus callosum | ll | 2.5%GA | 60 | 0.0881₂ | 0.0314₂ | 1 | 1 | 7 | 0.0004₂ | 0.8231₂ | |
| 2C₁ | Mouse | Cuprizone 5 weeks | Corpus callosum | mm | 2.%PFA/2.5%GA | 2.%PFA/2.5%GA [overnight] | 12 | 1 | 1 | 1 | ||||
| 2D₁ | Mouse | Cuprizone 6 weeks | Corpus callosum | ll | 2.5%GA | 48 | 0.1037₂ | 0.0040₂ | 1 | 1 | 3 | 0.2674₂ | <0.0001₂ | |
| 2D₁ | Mouse | Cuprizone 6 weeks | Corpus callosum | mm | 2.%PFA/2.5%GA | 2.%PFA/2.5%GA [overnight] | 29 | 1 | 1 | 1 | ||||
| 3A₁ | Afg3L2 | Ctrl | Spinal cord | jj | 2%GA | 2%GA/1% OsO4 | 14 | 0.1003₂ | 0.9153₂ | 1 | 1 | 2 | 0.2460₂ | 0.0033₂ |
| 3A₁ | OPA1 | Ctrl | Optic | hh | 4%PFA/5%GA | OsO4 | 10 | 1 | 1 | 1 | ||||
| 3A₁ | OPA1 | Ctrl | Optic | gg | 2.5%GA | 10 | 1 | 1 | 1 | |||||
| 3A | Plp1 | Ctrl | Spinal cord | ff | 2%PFA/2%GA | 26 | 0.1381 | 0.1172 | 1 | 1 | 1 | 0.6698 | <0.0001 | |
| 3A | OL:mtPstI | Ctrl | Spinal cord | ii | 4%PFA | 2%GA/100mM Suc [o.n.], 2%OsO4 [1 hr] | 43 | 0.0646 | 0.2539 | 1 | 1 | 3 | 0.2025 | 0.0086 |
| 3A₁ | MFN2 | Ctrl | Spinal cord | kk | 2%PFA/2%GA | 2%PFA/2%GA [2 hr], OsO4 [4 hr] | 13 | 0.0279₂ | 0.3607₂ | 2 | 1 | 4 | 0.2535₂ | 0.0074₂ |
| 3A₁ | MFN2 | Ctrl | Spinal cord | kk | 2%PFA/2%GA | 2%PFA/2%GA [2 hr], OsO4 [4 hr] | 14 | 2 | 1 | 3 | ||||
| 3B₁ | OPA1 | KO | Optic | gg | 2.5%GA | 9 | 0.0465₂ | 0.1136₂ | 1 | 1 | 1 | 0.0185₂ | 0.2759₂ | |
| 3B₁ | OPA1 | KO | Optic | hh | 4%PFA/5%GA | OsO4 | 15 | 1 | 1 | 1 | ||||
| 3B₁ | Afg3L2 | KO | Spinal cord | jj | 2%GA | 2%GA/1% OsO4 | 23 | 1 | 1 | 1 | ||||
| 3C₁ | MFN2 | KO | Spinal cord | kk | 2%PFA/2%GA | 2%PFA/2%GA [2 hr], OsO4 [4 hr] | 29 | 0.0207₂ | 0.3888₂ | 2 | 1 | 5 | 0.0054₂ | 0.6450₂ |
| 3C₁ | MFN2 | KO | Spinal cord | kk | 2%PFA/2%GA | 2%PFA/2%GA [2 hr], OsO4 [4 hr] | 41 | 2 | 1 | 4 | ||||
| 3C | OL:mtPstI | KO | Spinal cord | ii | 4%PFA | 2%GA/100 mM Suc [o.n.], 2%OsO4 [1 hr] | 40 | 0.0136 | 0.4671 | 1 | 1 | 2 | 0.0056 | 0.6319 |
| 3D | Plp1 | KO | Spinal cord | ff | 2%PFA/2%GA | 46 | 0.1101 | 0.9577 | 1 | 1 | 1 | 0.4990 | <0.0001 | |
₁TEM images from multiple publications have been pooled to increase “N”. ₂r 2 and p value for the individual dataset included in every figure. When N < 30, r 2 and p value is given for all the pooled data. ₃Specimen protocol. [aa] Perfusion/Fixation: 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1M PBS. Rinsed with 0.1 PBS and postfixed in 2% OsO4 in 0.1M PBS at +4°C for 2 hr Dehydrated in 70%‐OH for 30 min +4°C, 95%‐OH for 30 min +4°C, 100%‐OH 20 min RT, Acetone 2 × 15 min RT, LX‐112/Acetone (1:2) 4 hr RT, LX‐112/Acetone 1:1 overnight RT, LX‐112/Acetone (2:1), overnight RT, LX‐112 overnight RT. Embedding: LX‐112 at +60°C. [bb] Perfusion: 0.9% NaCl and 0.5 ml/L of heparin. Fixation: 2% paraformaldehyde and 2% glutaraldehyde solution. Sectioning: 50 nm. [cc] Perfusion/Fixation: Modified Karnovsky fixative. Embedding: plastic. Sectioning: ultrathin. Imaging: Philips CM120 transmission electron microscope. [dd] Perfusion/Fixation: 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M PBS for 1 week. Embedding: Rinsed in PBS, 1% osmium tetroxide, stained with uranyl acetate for 1 hr, dehydrated in a series of graded alcohols, and embedded in Araldit epoxy resin. Sectioning: Ultrathin sections. Imaging: Philips EM420. [ee] Perfusion: Ringer solution containing 100,000 IU/l Heparin. Fixation: Modified Karnovsky fixative: 2% formalin, 2.5% glutaraldehyde (Axonlab) in 0.1 M phosphate buffer, and 70 mM calcium chloride (Sigma). Postfixed 4–10 days in 4% formalin. Sectioning: 100 nm, with toluidine blue for light microscopy or kept unstained for electron microscopy. Imaging: Zeiss 10. [ff] Perfusion: PBS: Fixation: 2% paraformaldehyde and 2% glutaraldehyde solution. Embedding: resin‐embedded. Sectioning: Ultrathin. [gg] Perfusion: 0.1M PBS. Fixation: 2.5% glutaraldehyde in 0.1M, pH 7.3 in PBS. Sectioning: 85nm, collected and stained with uranyl acetate (1.5% in EtOH 70%). Imaging: Hitachi 7100. [hh] Perfusion: PBS. Fixation: 4% paraformaldehyde and 5% glutaraldehyde in cacodylate buffer. Postfixultraed in osmium tetroxide, dehydrated in acetone. Embedding: epoxy resin. Sectioning: Ultrathinsections stained with uranyl acetate and lead citrate. Imaging: Philips CM1000. [ii] Animal perfusion: 0.1M PBS Fixation: 4% paraformaldehyde in 0.1M PBS, postfixed overnight in 2% glutaraldehyde plus 100mM sucrose in 0.15M phosphate buffer before incubation with 2% OsO4 for 1 hr. Dehydration in graded ethanol solutions. Embedding: epoxy resin. Sectioning: 60–90 nm. Imaging: Philips CM10. [jj] Perfusion: PBS. Fixation: Postfixed in 2% glutaraldehyde in 0.12M PBS, 1% osmium tetroxide, dehydration with ethanol and propylene oxide. Embedding: Epon. Sectioning: 70 nm stained with uranyl acetate and lead citrate. Imaging Phillips CM10. [kk] Animal perfusion: 2% paraformaldehyde, 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.3) for 5min. Fixation: 2% paraformaldehyde, 2% glutaraldehyde, 0.1M cacodylate buffer at pH 7.3 for 2 hr at 4C, and washed in 0.1M cacodylate buffer, and osmicated for 4 hr in 1% OsO4. Embedding: epoxy 812‐Araldite. Sectioning: Ultrathin sections collected on cellodin‐coated single slot grids and stained with uranyl acetate and lead citrate. Imaging: Technai G2 electron microscope. [II] Perfusion: 0.1 M PBS. Immersed with 2.5% glutaraldehyde TEM fixation solution (Servicebio) and 1% osmic acid in 0.1 M PBS (pH 7.4). Dehydration with ethanol, and embedded with acetone and embedding medium (Servicebio). Sectioning: ultrathin (Leica UC7). Imaging: FEI Tecnai G2 20 TWIN. [mm] Perfusion: 2% paraformaldehyde, 2.5% glutaraldehyde postfix overnight. Washed in 0.1M sodium cacodylate buffer. Stained with 1% osmium tetroxide and 0.5%ptassium ferrocyanide in 0.5% sucrose for 1.5 hr. Dehydraded in alcohol and embedded in epoxy resin. Sectioning: Ultrathin. Imaginge: FEI Morgagni 268 TEM.