Paraformaldehyde stock

Paraformaldehyde Stock (see Note 4) Heat 800 mL of distilled H20 to 55° C. Add 80 g of paraformaldehyde prill type and stir continuously on heat/stir plate. Do not allow powder to settle. Do not allow temp to exceed 60° C (see Note 5). Stir solution for 15 min at 55° C. Add a few drops of 1 N NaOH until the solution just clears. A small amount of flocculent material may remain. Filter through Whatman No. 1 filter paper. Bring volume to 1 L and store at 4° C for up to 1 month. 

Preparing and using fixatives containing formaldehyde must be done in a fume hood. 1. Paraformaldehyde Stock Solution (15%) makes 200 ml... 

Always check pH after all components mixed. The paraformaldehyde stock solution added will always increase pH variably. A pH-meter electrode dedicated to pH measurement of fixative is recommended. 

The method for the demonstration of AChE followed the procedures of Koelle and Friedenwald (1949) and Lewis (1961). Slides were incubated for 15 h in 100 mL of stock solution (see below) to which had been added 116 mg of S-acetylthiocholine iodide and 3.0 mg ethopropazine (May Baker). The slides were rinsed with tap water and developed for 10 min in 1% sodium sulphide (1.0 g in 100 mL of water) at pH 7.5. They were then rinsed with water and immersed in 4% paraformaldehyde in phosphate buffer for... 

Paraformaldehyde/Lysine/Periodate Fixative (PLP ) (2) (see Note 6) Place 30 mL of lysine stock in a 50 mL screw cap tube. Add 10 mL 8% paraformaldehyde. Add 0.085 g of Sodium w-periodate powder. Shake until powder dissolves. 

Paraformaldehyde (16%), individual vials (Electron Microscopy Sciences). Dilute to 4% in lx PBS (from lOx PBS stock) for fixation. 

Stock formaldehyde solution (25%) see Note 4) Dissolve 10 g paraformaldehyde in 30 mL water (in capped 50 mL tube). Heat to 75 °C in water bath with frequent mixing. Add lOA NaOH to clear (usually 5-20 drops). Add H2O to 40 mL. Store in 1-mL aliquots at -20 °C. 

Nuclear-associated vesicles (either chromatin-bound or fused into an envelope) can be labeled. Alternatively, vesicles can be prelabeled prior to nuclear assembly reactions. To label chromatin-associated vesicles, a 10-/il extract containing nuclei is mixed with 10 ju.1 of DiOC6 or DilCis, each at 20 /xg/ml in egg lysis buffer (stock B), and incubated at room temperature for 20 min. Samples are visualized under the fluorescence microscope using the appropriate filter sets. Nuclear DNA can be simultaneously labeled at a final concentration of 0.1 /u.g/ml Hoechst 33342. If desired, samples can be fixed and stained simultaneously. A IO-/1I sample is mixed with 10 /x.1 of 7% paraformaldehyde containing 20 ju.g/ml of either of the lipophilic dyes. 

Paraformaldehyde, 0.2% glutaraldehyde m PBT (see above). Make this solution from a frozen, E. M. grade 25% glutaraldehyde stock (Sigma Grade 1) and freshly prepared paraformaldehyde. 

Cells were washed with prewarmed PBS (Fluka Chemicals, Buchs, Switzerland) and fixed in a 4% paraformaldehyde solution (freshly prepared from a 20% stock solution, paraformaldehyde powder from Fluka Chemicals, Buchs, Switzerland). After rinsing with PBS, the cells were penneabilized using 0.5% Triton X-100 (Fluka Chemicals, Buchs, Switzerland). The substrata were incubated with Alexa Fluor 488 phalloidin (1 100 dilution in PBS Invitrogen AG, Basel Switzerland) for 25 min at room temperature and rinsed with PBS. The nucleus was stained with DAPI (1 1000 dilution in PBS Invitrogen, Basel, Switzerland) for 15 min at room temperature. The substrata were rinsed again with PBS and kept in PBS in order not to dry off. 

Prepare 400 ml of Ix Robb s Medium from the 5x stock containing 4% paraformaldehyde. Warm to 30 C. Use as a freshly prepared solution. 

Do not use commercial formaldehyde for histological preparations as it is unstable. Prepare formaldehyde from paraformaldehyde and store as a 20% stock at 4 C for 1-2 weeks, depending on the preparation. 

To make a 20% stock solution, add 200 mg of EM-grade paraformaldehyde per milliliter of HjO and heat at 60°C on a stir plate (in a ventilated chemical fume hood) to dissolve. Add a trace of NaOH to help dissolve the paraformaldehyde (no more than 1 ml of 1 N NaOH to 100 ml of HjO). Do not overheat if the solution boils or turns brown, discard and start over. Adjust pH with 1 N HCl (pH should be 7.0). formaldehyde is typically diluted to 4% in the desired buffer. 

A 20% stock of formaldehyde lasts up to 1 week if kept at 4°C. Important Do not use a commercially available 37% stock formaldehyde rather, make a 20% stock solution from paraformaldehyde (see recipe provided in Protocol 12.1). 

Prepare a fresh stock solution of 8% paraformaldehyde as follows Add 8 g of paraformaldehyde to 100 ml of H O. Place on a hot plate at high setting in a chemical fume hood. Heat with stirring until steaming, but do not boil. Add 5-7 drops (may take a few more or less) of 1 N NaOH, one drop at a time until paraformadehyde goes into solution. 

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