MicroscopyProtocols
p-Phenylenediamine: An Adjunct to and a Substitute for Osmium Tetroxide
E. Ann Ellis Microscopy & Imaging Center, Texas A&M University, College Station, TX
ellisa@mic.tamu.edu
p-Phenylenediamine (PPD) has been used to retard
photobleaching in fluorescence microscopy, to increase the intensity of osmium staining [1, 2], and to improve lipid preservation [3, 4]. Phend et al. [5] used PPD to improve post-embedding immunolocalization in epoxy-embedded specimens; more recently, Brorson et al. [6] recommended including 1% (wt/vol) PPD in all dehydration steps for post-embedding immunolocalization. Experience with a number of animal tissues and microorganisms has resulted in the following protocol, which has been used to improve post-embedding immunolocalization in specimens embedded in either epoxy resins or acrylic resins such as LR White and LR Gold. 1. Fixation: In a properly functioning fume hood, fix
specimens for 1 hour in freshly prepared 4% (wt/vol) parafor- maldehyde-0.5% (vol/vol) glutaraldehyde in 0.1 M PIPES or HEPES buffer, pH 7.2–7.4 at room temperature. For microor- ganisms, 2.5–5% (vol/vol) acrolein in the same buffers or growth medium can be used. (Safety alert: If acrolein (tear gas) is used, potassium disulfite should be on hand to neutralize the used fixative and for use if there are accidental spills.) All subsequent steps until 100% ethanol (ETOH) should be done in an ice bath on a shaker with slow agitation. 2. Buffer Washes: Wash the specimens 4 × 15 minutes
with cold (on an ice bath) 0.1 M buffer (the same buffer as used above) containing 0.5–1.0% (vol/vol) dimethylsulfox- ide (DMSO). Te specimen can then be kept in cold buffer without DMSO in the refrigerator overnight if necessary. Wash specimens 2 × 15 minutes in cold buffer plus 0.1 M glycine to remove any unbound aldehydes. Ten rinse with cold buffer without glycine for 15 minutes. 3. Tannic Acid Treatment: Incubate the specimens for
one hour in 1% (wt/vol) tannic acid in 0.1 M sodium maleate buffer, pH 6.0, followed by two quick rinses with 0.1 M sodium maleate buffer. Some protocols use 1% (wt/vol) tannic acid in the primary fixative, and thus the incubation in tannic acid in sodium maleate buffer is not necessary. 4. Dehydration: Dehydrate in 40% and 60% (vol/vol)
ETOH that contains 1% PPD (wt/vol) for 15 minutes each and then incubate overnight in freshly prepared 1% (wt/vol) PPD in 70% ethanol. Continue the next day with further dehydra- tion in 80% and 95% ethanol that contains 1% (wt/vol) PPD. Dehydration can be stopped here or continued to 100% ethanol containing 1% PPD. 5. Resin Infiltration: Specimens can then be infil- trated with the appropriate embedding medium. If epoxy
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resins are used and propylene oxide is used as the transi- tional solvent, 1% (wt/vol) PPD should be included in all propylene oxide containing steps. Low-viscosity epoxy resins based on the corrected Spurr formulation [7] or any straight-chain epoxy-resin formulation have been used successfully. Epoxy resins containing Araldites 502, 506, or 6005 are not recommended because the labeling efficiency is still very poor as a result of the benzene ring structure of these epoxies. If LR White acrylic resin is used, infiltra- tion should be continued on an ice bath on a shaker for 30 minutes for each infiltration step (2 parts 95% ETOH + PPD: 1 part LR White; 1 part 95% ETOH + PPD: 1 part LR White; 1 part 95% ETOH + PPD: 2 parts LR White; three changes of pure LR White for 30 minutes each followed by a fourth change overnight). The usual precautions to exclude oxygen from the acrylic embedding medium by bubbling dry nitrogen or argon through an aliquot of the embedding medium should be done to ensure uniform polymerization of the specimen block. PPD (Sigma catalog No. P-6001), DMSO, and sodium
maleate were all purchased from Sigma, St. Louis, MO. (Safety alert: PPD is potentially allergenic and care should be taken to avoid skin or other contact with this compound.)
Figure 1: Immunolocalization of chemotaxis protein tsr in Eschericia coli with 12-nm colloidal gold (arrows) labeled secondary antibodies. Scale bar equals 200 nm.
doi:10.1017/S1551929510000908
www.microscopy-today.com • 2010 September
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