Preparation of periodatelysine paraformaldehyde for perfusion of mouse tissue3

Reagents

* Lysine monohydrochloride (Sigma): dissolve 6.85 g in 187.5 ml dH20

- Sorensen's salt (Na2HP0„.2H20) (Sigma): dissolve 0.9 g in 50 ml dH20

• Paraformaldehyde (BDH): dissolve 10 g of paraformaldehyde in 100 ml dH20 and ten drops of 1 M NaOH at 56°C, then cool to room temperature"

Protocol 6 continued

• Sodium periodate (Sigma)

• 0.1 M phosphate buffer: 7,8 g NaH2PO„.2HiO (BDH) dissolved in 250 ml dHjO and 34 g Na2HP04.2H20 (BDH) dissolved in 1 litre dH,0. Mix the dissolved NaH2P0„.2H20 and Na2HP04.2Hi0 and dilute 1:1 to make the 0.1 M working solution.

Method

1 Buffer the 187.5 ml lysine monohydrochloride with the 50 ml Sorensen's salt to give apH of 7.4.

2 Mix the buffered lysine monohydrochloride and the paraformaldehyde.

3 Make the volume to 500 ml with 0.1 M phosphate buffer.

4 Add 1.07 g of sodium periodate to the lysine/paraformaldehyde.c a Adapted from ref. 11.

bThis paraformaldehyde solution must not be confused with the solution described in Protocol 3, ' The pH will fall to about 6.4-6.6 on mixing. This appears not to have an adverse effect on antigen preservation, but tissue preservation may not be ideal. Some variation in pH can be found at this point and improved preservation is obtained by bringing the pH to about 6.8 using a few drops of 20% NaOH.

• Butterfly needle

• Container for organs

Protocol 7

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  • Niamh
    Is sodium periodate soluble in PBS buffer?
    8 years ago

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