Staining and Embedding the Whole Mouse Brain for Electron Microscopy

Transcription

1 Staining and Embedding the Whole Mouse Brain for Electron Microscopy Shawn Mikula, Jonas Binding, Winfried Denk Supplementary Item Supplementary Figure 1 Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Supplementary Protocol Title or Caption Comparison of white matter in hemispheres stained using different longincubation (24 hr) protocols Staining protocols Outcomes for various staining protocols Nodal and internodal error rates for white and gray matter at different resolutions Step-by-step whole-brain sample preparation protocol

2 Supplementary Figure 1 Supplementary Figure 1. Comparison of white matter in hemispheres stained using different long-incubation (24 hr) protocols. (a) Osmium tetroxide (Os) incubation. (b) Osmium tetroxide incubation followed by uranyl-acetate enhancement (Os->UA). (c) Osmium tetroxide, followed by thiocarbohydrazide, and then by a second osmium tetroxide incubation (OTO). (d) Periodic acid, followed by thiocarbohydrazide, and then by osmium tetroxide (wbpatco). See text for details. Insets: magnified sub-regions.

3 Supplementary Table 1 incubation steps references staining protocol primary secondary tertiary solution temp solution temp solution temp ( C) ( C) ( C) Os 80 mm OsO Os -> PbAsp 80 mm OsO 4 20 lead aspartate Os -> UA 80 mm OsO mm uranyl acetate wbpatco 90 mm periodic acid, mm mm M cacodylate, ph TCH, 0.1M OsO cacodylate, ph 7.4 OTO 80 mm OsO mm TCH mm OsO 4 roto 80 mm OsO 4, mm TCH mm 20 35,36 72 mm ferrocyanide, OsO M cacodylate, ph 7.4 Supplementary Table 1. Staining protocols. All staining steps use ddh 2 O as solvent.

4 Supplementary Table 2 small samples, 90 min incubations hemispheres or whole brains, hr incubations staining membrane myelin staining tissue membrane myelin staining tissue protocol contrast contrast uniformity preservation contrast contrast uniformity preservation Os Os -> PbAsp Os -> UA PATCO OTO roto Supplementary Table 2. Outcomes for various staining protocols, ranging from very strong (+++) to very weak (-).

5 Supplementary Table 3 white matter nodes of Ranvier inter-node segments total errors/mm resolution (nm) errors/node (%) errors/mm errors/segment (%) errors/mm gray matter Supplementary Table 3. Nodal and internodal error rates for white and gray matter at different resolutions.

6 Supplementary Protocol Materials Reagents Sample Adult whole mouse brain. Staining and fixation Isoflurane (Baxter) Cacodylate buffer (Serva) Acrolein (Sigma) Periodic acid (Sigma) Osmium tetroxide (Serva) TCH (Sigma, cat. no. T-2137) Dehydration and infiltration Water (double-distilled) Acetone (Sigma) Embedding o ERL-4221 (Serva) o DER 736 (Serva) o NSA (Electron Microscopy Sciences) o DMAE (Serva) Or o o o Quetol 651 (Electron Microscopy Sciences) NSA (Electron Microscopy Sciences) DMAE (Serva) Equipment Perfusion #2 forceps (Fine Science Tools) Coarse and fine scissors (Fine Science Tools) Nitrile gloves Plastic tray (generic)

7 Hemostat (Fine Science Tools) Serrated curved forceps (Moria MC31, Fine Science Tools) 22-gauge cannula with syringe attachment (use a needle and remove the sharp tip) Aneurysm clamp (Fine Science Tools) 40 ml syringe Transfer pipet (generic) Razor blade (VWR) 50 ml centrifuge tubes Lab rocker (VWR) EM processing and embedding Centrifuge tubes (Sarstedt) Embedding oven Custom made silicone molds with dimensions 7 mm x 10 mm x 18 mm Block trimming Leica EM trimmer Reagent setup Buffer Add 4.28 g sodium cacodylate per 50 ml water, and bring volume up to 100 ml with water, to get a final concentration of 0.2 M sodium cacodylate buffer. Adjust the ph to 7.4 with hydrochloric acid and store in the fridge. Add equal parts water to make up 0.1 M buffer as needed. Fixative Add 20% acrolein to 50 ml of 0.2 M sodium cacodylate buffer and bring volume up to 100 ml with water. Osmium tetroxide Periodic acid Add 90 mm periodic acid to 0.1 M cacodylate buffer, as prepared above. Thiocarbohydrazide Add 100 mm thiocarbohydrazide to 0.1M cacodylate buffer, as prepared above. Procedure Fixation by perfusion -- Time: < 5 min After anesthesia, place the mouse supine in a plastic tray and make a horizontal cut into the upper abdomen. Cut into the diaphragm, then cut the ribs laterally. Fold the chest flap back using a hemostat. The heart should then be exposed with visible auricles. Hold the heart in place gently using serrated, curved forceps. Insert a 22-gauge cannula attached to the syringe into the left ventricle and secure it in

8 place using an aneurysm clamp. Pierce or cut the right auricle using a needle or a pair of fine scissors. Begin perfusing the mouse using the syringe at a rate of 0.5 ml / sec with 40 ml of a 0.418M (10%) solution of acrolein in 0.1M sodiuim cacodylate buffer. When the fixative has all flowed through the mouse, carefully dissect out the brain using scissors and forceps. Proceed to the post-fixation step. Dissection Time: 10 min Decapitate the mouse and remove the muscle and skin tissue around the cranium and cervical vertebrae with small scissors and forceps. Remove skull bone with #2 forceps beginning at the back of the skull and moving rostrally until the entire surface of the brain is exposed. Periodically drop fixative solution onto the surface of the brain using a transfer pipet to prevent tissue dehydration and to facilitate quick fixation. Invert the brain and, using small scissors, cut the cranial nerves to separate the brain from the remaining skull tissue. Post-fixation and rinses -- Time: 88 hrs Place the brain into 50 ml of acrolein solution (the same fixative used in the transcardial perfusion) for post-fixation for 48 hrs at 2 C. Rinse the brain five times for eight hours using 50 ml of 0.1M cacodylate buffer. If the brain is to be used for small samples, dice the brain into small cubes using a razor blade during the rinses. For the procedure using small samples, shorten the staining times according to the manuscript. Following is the procedure for whole brain staining. Staining Time: 208 hrs (between 8 and 9 days) 1. Place the whole mouse brain in a 50 ml tube containing 25 ml of 90 mm periodic acid in 0.1M cacodylate buffer for 48 hrs at room temperature. Gently agitate by placing the tube on a laboratory rocker on a slow speed. 2. Rinse the brain four times for eight hours each in 50ml of caocdylate buffer at room temperature, as prepared above. 3. Transfer the brain to a fresh container and, for 48 hrs, immerse it in a solution of 100 mm thiocarbohydrazide in 0.1 M cacodylate buffer at 50 C. 4. Rinse the brain again four times in buffer for eight hours each and change containers. 5. Immerse the brain in a solution of 2% osmium tetroxide in water at room temperature for 48 hrs. 6. Proceed to the embedding step. Embedding Time: 120 hrs (5 days) Dehydration Step the tissue through a series of water / acetone mixtures (50%, 75%, 100%, 100% acetone) of 50 ml each, with each step lasting 12 hrs at room temperature. Infiltration Infiltrate the epoxy resin with 12 hr steps of 25 ml each (50%, 100%, 100% epoxy) with gentle agitation on a lab rocker at room temperature. For Spurr s resin: 10g ERL g DER g NSA

9 0.2g DMAE For Quetol resin: 10g Quetol g NSA 0.15g DMAE Place the brain into a custom made silicone mold and cure in a laboratory oven at 70 C for at least 24 hrs. When the block is hard, trim it according to desired dimensions with the EM Leica trimmer.