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Supplementary Data  to

Buzgo M, AS Chanderbali, S Kim, Z Zheng, D Oppenheimer, PS Soltis, DE Soltis  2007  
Floral developmental morphology of Persea americana (avocado, Lauraceae): the oddities of male organ identity. International Journal of Plant Sciences (in press)

Table of Content

Fixation and dehydration

Material

• Ethanol (EtOH)
• Distilled H₂O, or DEPC-treated H₂O (H₂O_depc, for in situ hybridizations)
• Tissue samples: fixed (e.g., FAA, PFA), transferred and washed in 70 % EtOH several times

Fixation

FAA (Formalin, Acetic acid, Alcohol)

Good for histology and in situ hybridization.

Mix: 
(Ruzin, S.E.  1999  Plant Microtechnique and Microscopy.  Oxford: University Press. pp. 322. ISBN: 0‑19‑508956‑1)

Can be stored several days in dark and cool.

• Drop samples into FAA 
• Apply vaccum for few seconds (20-60) several times in fast succession (3-6x) with careful release in between.
• Incubate for 4-24 hours (depending on size, postfixation possible).
• Transfer into EtOH 50 %
• Transfer into EtOH 70 % for storage

PFA (Paraformaldehyde 4%)

Particularly for in situ hybridization.

Mix in beaker & stirrer

• Cool the fixation solution on ice
• Drop samples into PFA 
• Apply vaccum for few seconds (20-60) several times in fast succession (3-6x) with careful release in between.
• Incubate for 4-24 hours (depending on size, postfixation possible).
• Transfer into EtOH 50 %
• Transfer into EtOH 70 % for storage

Dehydration series

• EtOH             70 %
• EtOH           85 %
• EtOH           95 %
• EtOH         100 %

Samples can be stored

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Paraplast embedding

Material

• Paraplast™ or Paraplast Plus™ Tissue Embedding Medium [#23-021399 and #23-021400, respectively, Fisher Scientific Com. L.L.C.; Houston, TX 77038, USA]. Paraplast Plus™ contains DMSO for rapid infiltration).
  Melt Paraplast in advance (56°C, takes pretty long)
• Incubator or oven with steady temperature (56°C)
• Histoclear® (simple histology) [#HS3200/EA, National Diagnostics, Atlanta, GA 30336, USA], Xylenes (for in situ hybridizations)
• Embedding molds: commercial device. Or form molds: deep enough but ≤1cm, wide enough from cardboard, aluminum foil, plastic lid, small petri-dish, etc.
• Optional: warm bench and glycerol
• Flame (gas or spiritus), dissection needles and forceps
• Bowl with ice water or cool water
• Firm razor blades
• Wood blocks or manufactured microtome sockets.
• Fixed samples, dehydrated to 100% EtOH according to Dehydration above.

Infiltration

• Cover samples with Xylene or Histoclear 100 %
• Fill up with molten Paraplast, let melt again at 56°C
• Replace with molten Paraplast, let melt again at 56°C
• Replace with molten Paraplast, let melt at 56°C and let all Xylene evaporate (no more scent is detectable).

• EtOH         100 % +0.01% Eosine (dye that increases sample visibility when embedded in Paraplast)
• EtOH:Xylene   2:1
• EtOH:Xylene   1:1
• EtOH:Xylene   1:2
• Xylene        100 %     (samples can be stored)
• Xylene        100 %     (samples can be stored)

Embedding

• Lubricate inner surface of mold with Glycerol: may help removal of wax block from after hardening
• Pour Paraplast and sample swiftly into embedding mold, fill up with molten Paraplast if necessary
• Using the flame-warmed needles & forceps: place samples in appropriate position while Paraplast hardens.  If placement goes awry, if too many air bubble occur, if added Paraplast forms separate layer, re-melt tub in stove (on a petridish to avoid spilling and leaking).
• Let Paraplast go somewhat firm ("skin").
• Drop mold into ice water till cold
• Store until use

Block

• Remove samples from mold
• Cut and trim with firm razor blade to appropriate shape
• Weld sample onto wood block: flame-warm blade and streak between wood block and sample bottom.
• Let cool completely
• Trim sample to trapezoid shape and remove excessive Paraplast

Continue with Microtome sectioning using rotation microtome (10-5 µm).

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Metacrylate resin embedding

Replacing Kulzer Technovit H8100 for about half the price.

Material

Metacrylate Mix

• Butyl methacrylate [Sigma-Aldrich #23,586-5]      80ml
• Methyl methacrylate [Sigma-Aldrich #64200]       20ml
• Benzoin ethyl ether  [Sigma-Aldrich #17,200-6]   0.5g
• DTT (reductant)                               10mM (=0.154g)
• Gas removal:
  Gently bubble N₂ through mixture for ~30min (get rid of O₂).
  Use an 250ml Erlenmeyer with double-hole plug, one hole with plastic pipette tip as regulation walve, one hole for plastic tube with pasteur pipette reaching into mix, put Erlenmeyer into a styrofoam box, put liquid N₂ into Erlenmeyer, plug it, regulate by opening of styrofoam box and plastic pipette tip. 
• Store in darkness at -20°C

Other material

• Ethanol (EtOH)
• Distilled H₂O, or DEPC-treated H₂O (H₂O_depc, for in situ hybridizations)
• Optional: DTT dissolved in EtOH (DTT 1mM for up to EtOH 95%, 10mM for 100%)
• Embedding molds: commercial device, or form molds: deep enough but ≤1cm, wide enough from aluminum foil, plastic lid, small petri-dish etc.
• Optional: long wave UV source (~420nm)
• Firm razor blades
• Improvised or manufactured microtome sockets (preferably plastic or aluminum).
• Bi-component epoxy-resin or similar glue (Araldite, Loctite Quick Set™ Epoxy, etc.)
• Fixed samples, dehydrated to 100% EtOH according to Dehydration above (optional with DTT, 1mM for up to 95%, 10mM for 100%).

Embedding: infiltration and polymerization

  Then incubate in

• 2:1 EtOH:Metacrylate mix 30min, -20°C
• 1:1 EtOH:Metacrylate mix 30min, -20°C
• 1:2 EtOH:Metacrylate mix 30min, -20°C
• 100% Metacrylate mix overnight -20°C (dark), one day 4°C
• 100% Metacrylate mix 1h, -20°C
• 100% Metacrylate mix 2h, -20°C
• 100% Metacrylate mix, evacuate on 10min at 4°C, leave in hood overnight (dark)
   
• 100% Metacrylate mix exchange, seal and keep air completely out (O₂ inhibits polymerization)
• Expose to long wave UV (~420nm) for up to 6h at 4°C, or on ice, to fluorescent ceiling light or to sunlight behind a window.
• Samples can be stored

Block

• Remove hardened block with included sample from mold
• Trim to appropriate size and easy positioning with firm blade
• Glue to microtome socket using bi-component epoxy-resin glue
• Let harden

Continue with Microtome sectioning using rotation microtome (≤5 µm) and microscope glass slides (clean, untreated)

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by Matyas Buzgo, Department of Botany, University of Florida, Gainesville, FL 32611, USA
This page was last updated October 3, 2007.