Poly-L-lysine coated slides

Poly-L-lysine coating improves adherence of tissue sections to the glass slides. This is important for immunolocalization, where the tissue on the slide undergoes lengthy and sometimes harsh treatment and thus tends to be lost during the process.

Reagents/Supplies: Sigma P8920 Poly-L-Lysine solution (0.1% w/v) Diluted 1:10 in distilled or ultrafiltered water to make working solution, store refrigerated in a plastic container. Use for no more than about 25 slides per 50 ml of the working solution. Slides: VWR Micro slides 4830-036 (other brands and flavors may be OK, but among those we tested the above worked the best).

• Clean slides in acetone for 5 minutes.
• Remove slides and wipe with a kimwipe (wear gloves).
• Air dry slides at an angle (leaning on a tube rack or other suitable support) on paper towels.
• Dip slides in 1:10 diluted Poly-L-Lysine solution in a plastic coplin jar for 15 minutes.
• Air-dry slides at an angle on paper towels.
• Mark the top right corner with a diamond edged pen.
• Bake the slides for 1 hour at 55°C.

Gelatin-subbed microscope slides

To improve adherence of semi-thin resin section to glass slides for staining, use the following procedure:

• Make 0.5 % w/v gelatin (Knox, the grocery store jello stuff) in distilled/ultrafiltered water. Dissolve the gelatine while stirring on a heated stir plate (low heat setting)
• In the meantime, etch pre-cleaned frosted microscope slides in 5% H2SO4 for 5 min, stirring occasionally with an applicator stick and making sure the slides are not stuck together.
• Wash well (many times) in distilled water, again stirring the slides. Keep the slides under water, do not allow to dry.
• Fill a Coplin jar with the gelatin solution and immerse the slides for a minute or so, then remove and place in a slide rack, let dry overnight.
• Store the slide in a storage box.

Gatenby's glue subbed microscope slides

To improve adherence of cryosections for subsequent immunostaining.

• Prepare Gatenby’s glue: 27% v/v ethanol, 6.3% v/v acetic acid, 1.35% w/v gelatin (Knox food-grade gelatin from the grocery store is OK), 0.09% w/v chrom alum (KCr(SO₄)₂). Stir without heating until the gelatin dissolves (may take several hours).
• Etch pre-cleaned frosted microscope slides in 5% H₂SO₄ for 5 min, stirring occasionally with an applicator stick and making sure the slides are not stuck together.
• Wash well (many times) in distilled water, again stirring the slides. Keep the slides under water, do not allow to dry.
• Apply a small drop of Gatenby’s glue on a slide and spread with a gloved finger. Alternatively, put a small drop of Gatenby’s glue close to one end of the slide and use the edge of another slide to drag and spread the glue across, towards the opposite end of the slide.  Let the slides dry, and store them in a storage box protected from dust. The shelf life of these subbed slides has not been established, but they should be OK for several days. 

Histochemical staining for β-glucuronidase (GUS) reporter in plants

This protocol has proven successful for many plant species and tissues. It contains several improvements over the original protocol and also deals with possible sources of errors and their elimination. Download: GUS_Localization_in_plants.pdf

Embedding tissues in low-melting polyester wax (Steedman's wax) for immunolabeling

Steedman's wax (Steedman, 1957) is a low melting point embedding medium for immunohistochemistry. It is used instead of the classical paraffin wax when preservation of antigenicity is important. Download a detailed protocol here: Steedman's wax.pdf 

Advantages:

• Soluble in ethanol, no need to use hazardous solvents such as xylene.
• Low melting point (~35 °C) allows embedding without excessive heating. Much more convenient to work with than paraffin.
• Excellent preservation of antigenicity.
• It is a ribboning medium, thus serial sectioning is possible.

Disadvantages:

• Low melting point. Embedded blocks should be kept at cool or refrigerated, sectioning requires well air-conditioned rooms. Shipping the embedded blocks may be problematic in summer.
• More expensive than paraffin (~ $40 per 1000g)
• Section adherence to slides may be weak for certain specimens. Problematic for in situ hybridization, where most sections may be lost during the lengthy protocol.

This embedding medium has been used for immunolocalization of numerous antigens in both plant and animal tissues. As always, optimal fixation and immunostaining conditions for a particular antigen and antibody will have to be tested.

References:

1. Vitha, S., Baluška, F., Jasik, J., Volkmann, D., and Barlow, P. Steedman's Wax for F-actin Visualization in Actin: a Dynamic Framework for Multiple Plant Cell Functions, Staiger, C.J., Baluška, F., Volkmann, D., and Barlow, P., Editors. 2000, Kluwer: Dordrecht, The Netherlands. p. 619-636.
2. Steedman, H.F. A new ribboning embedding medium for histology. Nature, 1957. 179: p. 1345.

Microwave-assisted fixation and resin embedding of plant roots

This protocol was developed by Ann Ellis for our clients in Plant Pathology who study parasitic nematodes. The protocol allows excellent penetration of fixatives, good structural preservation and infiltration of the specimens with the embedding resin. The embedded material can then be sectioned for either light or electron microscopy. A scientific-grade, cooled laboratory microwave (Pelco Biowave) is used throughout the fixation and dehydration to improve and accelerate the process. Nematodes, just like insects, present a challenge for fixation and embedding, because of their low permeability. This is circumvented by applying osmium-vapor prefixation. The fixation step itself includes acrolein, an often-neglected fixation agent that offers excellent penetration and fixation quality. Please note that for other specimen types this protocol may have to be modified. Download the protocol: Microwave_protocol.pdf

Outline of the protocol:
• Osmium vapor prefixation
• Aldehyde fixation, microwave assisted
• Osmium post-fixation, microwave assisted
• Washing, microwave assisted
• Dehydration in methanol, microwave assisted
• Resin infiltration
• Polymerization

Digital Photomicrography with Nikon Coolpix cameras

The relatively inexpensive Nikon Coolpix consumer digital cameras mounted on a microscope are being used successfully in many labs for routine documentation and to produce publication-quality images. The example below shows Arabidopsis leaf mesophyll cells and their chloroplasts. The image was taken using a Nikon Coolpix 4500 camera coupled with an adapter and mounted on an Olympus BH2 microscope with a 40x objective.

The Coolpix 995 (3-megapixel) or Coolpix 4500 (4 megapixel) are easily adapted to be mounted on a microscope via their 28mm filter thread. Both of these cameras are now discontinued, but still available on the used market. Adapters for newer cameras are also available, but the above two models are preferable because of the ease with which they are mounted to the microscope. The Coolpix 995 and 4500 cameras can be controlled from a computer via the USB or serial cables to reproducibly set the imaging parameters, namely the zoom of the camera lens (to achieve reproducible magnification). Alternatively, a remote shutter cable can be also used. Contact Stan Vitha if you are considering such setup in your lab. Check below for some relevant links.

Remote Control Driver and GUI for the Nikon Coolpix 990, 950, 880, 775, and 995 Digital Cameras, for Linux and Windows.

CoolpixPhotoMicmac. A newsgroup dedicated to the use of Coolpix cameras on a microscope. Links to software downloads.

Krinnicam. A remote shutter software for Nikon Coolpixes that run on Windows 2000/XP platforms. It features automatic image downloading through the USB connection.