Golgi impregnations

Slow Modified Golgi Method for Stomatopoda and other Eumalacostraca.

Rationale here is to get the fixed brains into osmium dichromate soon but starting with low
osmium concentration.

Day 1, late afternoon. Fix living thorax without cooling in standard dichromate-sucroseglutaraldehyde.
Dissect out midbrain and eyestalk neuropils immediately, removing sheaths.
Leave at room temperature until following morning (circa 12 hours).

Day 2. Wash in dichromate without sucrose. Separate the eyestalks from the mid brain.
Transfer the eyestalks to half strength standard dichromate-osmium tetroxide (99ml 2.5%
dichromate, 1ml 1% osmium tetroxide). The standard admixture is dilute 1:1 with 2.5%
potassium dichromate). Transfer the mid brain to full strength standard dichromate-osmium
tetroxide solution.
Leave at room temperature for 24 hours in the dark.

Day 3. Transfer eyestalks to full strength standard dichromate-osmium tetroxide (99ml 2.5%
dichromate, 1ml 1% osmium tetroxide). Transfer mid brain to double strength standard
dichromate-osmium solution.
Leave at room temperature for 24 hours in the dark.

Day 4. Wash in 2.4% potassium dichromate, three changes over 10 minutes.
Place in 5 volumes 2.5% potassium dichromate with 1 volume 25% glutaraldehyde (No
sucrose!).
Leave at room temperature for 24 hours in the dark.

Day 5. Place specimens one by one into 0.75% silver nitrate, swirl and transfer 2x into fresh
silver nitrate.
Leave at room temperature for 24 hours in the dark.

Day 6. Wash in distilled water, place into half strength standard dichromate-osmium tetroxide
(see day 2).
Leave at room temperature for 24 hours in the dark.

Day 7. Place specimens one by one into 0.75% silver nitrate, swirl and transfer 2x into fresh
silver nitrate.
Leave at room temperature for 24 hours in the dark.

Day 8, 9. Dehydrate and embed in Durcupan.

DEHYDRATION AND EMBEDDING IN DURCUPAN (Durcupan™ ACM set for 1 L embedding
mixture | Sigma-Aldrich)

• Dehydrate through ethanol series (50%, 70%, 90%, 96%, 100%) at 8-minute steps
• Incubate tissue in 100% propylene oxide (2 x 10 min)
• Leave tissue in 3:1 propylene oxide to Durcupan for 15 mins - 1 hours on shaker
• Leave tissue in 1:2 propylene oxide to Durcupan for 15 mins - 2 hours
• Leave tissue in 1:6 propylene oxide to Durcupan for 30 mins – overnight (the longer the
better, but it is not necessary to leave overnight if time is constrained)
• Move tissue to 100% Durcupan and leave on shaker for 2 hours (again time is lenient
here, although the longer the better)
• Embed tissue at the bottom of BEEM capsules with fresh Durcupan
• Polymerize Durcupan overnight at 65 degrees.
• Turn off oven and allow the plastic to gradually cool.
• Trim block in a trapezoid shape and section at 30 microns.

Durcupan Recipes:

Soft Mix (for Golgis)- 30-micron sections

1x                                3x                          4x
A: 10.8g                      32.4g                     43.2g
B: 8.9g                        26.7g                     35.6g
C: 0.5g                        1.5g                       2.0g
D: 1.9g                        5.7g                       7.6g

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