This protocol is an excerpt from Zhang et al, The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging, J. Vis. Exp. (2017).
Antibody staining of the C. elegans intestine
<ol>
	<li>Fixation
	<ol>
		<li>Take a clean glass slide and use poly-L-lysine to generate a thin film for worms to stick on. Place 30 &#181;L 0.1-0.2% poly-L-lysine on the slide and place a second slide on the poly-L-lysine drop to make a &#34;sandwich&#34;. Then rub the slides gently a few times to wet the entire surfaces of both and let them air dry for 30 min. Label the frosted side of the slides with pencil. 
		NOTE: 0.2% poly-L-lysine aliquots of 200 &#181;L were made by dissolving the powder in dH2O; this can be stored at -20 &#176;C. Use high molecular weight poly-L-lysine for improved sticking of the worms. The concentration of poly-L-lysine is also critical. Too low concentrations will not allow worms to stick but too high concentrations may generate fluorescence background signal. A too thick film may loosen up in its entirety.</li>
		<li>Place a flat metal block firmly on the bottom of a container (e.g. a polystyrene container) filled with liquid nitrogen. 
		NOTE: One can use dry ice instead, but liquid nitrogen keeps a metal block more stable on the container bottom and chills well.</li>
		<li>Collect worms either by washing them off their plates with M9 or pick different stage worms (either eggs, L1, L2, L3, or L4 larval stage worms) onto each slide. Typically, pick ~100 larvae and embryos or ~20 adults to each slide. Place 10 &#181;L washed off worms onto the middle of the slide or pick eggs/worms into 10 &#181;L M9 or 10 &#181;L 1x PBS (phosphate buffered saline).
		<ol>
			<li>Use a pipette to spread out large numbers of worms to avoid clumping. 
			Note: Mixed populations of washed off worms, due to crowding and different thickness of stages, do not stick well and are less effectively freeze-cracked (see below), therefore picking stage-specific worms (or synchronized populations) gives superior results. Larvae stick better than adults and more animals can be placed per slide.</li>
			<li>Before picking worms onto slides, transfer them to a Nematode Growth Medium (NGM) plate without OP50 bacteria. Excess bacteria adherent to the worms can also interfere with sticking. Take care that worms do not dry out.</li>
		</ol>
		</li>
		<li>Gently place (drop) a 22 mm &#215; 22 mm coverslip cross-ways on top of the collected worms such that its edges hang over on at least one side of the slide. Press straight down gently but firmly with one or two fingers on the coverslip. Avoid shearing that will damage tissue integrity.</li>
		<li>Immediately and gently transfer the slide to the metal block in liquid nitrogen and let it sit for about 5 min to freeze. Then &#34;flick off&#34; the coverslip in one swift move by using the overhanging edge. 
		NOTE: This step must be done decisively and while the slide is frozen to achieve &#34;cracking&#34; of the cuticle. Caution: Please follow the PPE (Personal Protection Equipment) guidelines when working with liquid nitrogen.</li>
		<li>Immerse the freeze-cracked slides into a methanol-filled glass Coplin jar for 5 min at -20 &#176;C. Then transfer to an acetone-filled glass Coplin jar for another 5 min at -20 &#176;C. 
		NOTE: Methanol and acetone should be stored in -20 &#176;C for at least 30 min before use. After fixation, slides can be stored at -20 &#176;C. 
		CAUTION: methanol and acetone are toxic.</li>
		<li>Remove slides from jar and let them air dry at room temperature (RT) before use.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Antibody staining</td><td></td><td></td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma</td><td>P5899</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A949SK-4</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>4448</td><td></td></tr><tr><td>Microscope coverslips (22x22-1)</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>M9 Medium</td><td></td><td></td><td>lab made</td></tr><tr><td>OP50 bacteria</td><td>CGC</td><td></td><td></td></tr></tbody></table>

freeze-cracking of nematodes: a method to expose interior worm tissues for staining

Source: Zhang, N., et al. <a href="https://www.jove.com/video/56100/the-c-elegans-intestine-as-model-for-intercellular-lumen">The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.</a> J. Vis. Exp. (2017).
This video describes freeze-cracking, a method to make C. elegans tissues accessible for antibody staining by disrupting the cuticle.

Freeze-Cracking of Nematodes: A Method to Expose Interior Worm Tissues for Staining

Source: Zhang, N., et al. The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the ...

- Transfer washed C.&#160;elegans&#160;in&#160;liquid onto a glass slide previously coated with poly-lysine, a compound that will help worm cuticles stick to the glass surface. Gently press another glass slide straight down with an edge overhanging the bottom slide. To immobilize the worms without damage, compress the animals avoiding any lateral motions. Then, place the glass slides with sandwiched worms on a metal block inside an insulated laboratory container filled with a freezing agent, such as liquid nitrogen or dry ice.
Metal is an effective temperature conductor ensuring the block surface is cold enough to rapidly freeze the worm&#39;s cuticles to both glass surfaces. Once frozen, swiftly separate the glass slides to pull apart or crack open the worm cuticles. Proceed with tissue fixation and staining as disruption of the low permeability cuticle allows chemical fixatives and staining antibodies to access the interior worm tissues. In the following protocol, we will see a detailed demonstration of the freeze-cracking technique and tissue fixation.
- First, prepare a slide to fix the worms. Pipette 30 microliters of poly-L-lysine onto a slide, and then, set a second slide onto the other and rub&#160;them together to spread the solution evenly onto both. Now, peel apart the two slides and let them air dry for 30 minutes.
Next, place a flat metal block into a container filled with liquid nitrogen. Wash the plates with M9 solution and transfer about 10 microliters of M9 containing the worms. Alternatively, pick the worms and add M9 to the slide.
Once the worms are transferred, gently drop a&#160;coverslip&#160;crossways onto them such that the coverslip overhangs one edge. Then, gently press the coverslip without making any lateral movements.
Now, immediately transfer the slide to the metal block in liquid nitrogen and let it freeze. After five minutes, flick off the coverslip using the overhanging edge. Do this swiftly to get the proper crack of the cuticle.
Next, immerse the slide in a methanol-filled glass Coplin jar at minus 20 degrees Celsius. After five minutes, transfer the slide to an acetone-filled glass Coplin jar for another five minutes at the same temperature. The slides can be stained directly or stored at minus 20 degrees Celsius.

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Caenorhabditis elegans (worm)

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2.9K Views. - Transfer washed C. elegans in liquid onto a glass slide previously coated with poly-lysine, a compound 

Freeze-Cracking of Nematodes: A Method to Expose Interior Worm Tissues for Staining

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Name	Company	Catalog Number	Comments
Antibody staining
poly-L-lysine	Sigma	P5899
Methanol	Fisher Scientific	A452-4
Acetone	Fisher Scientific	A949SK-4
Permount	Fisher Scientific	SP15-100
Microscope slides	Fisher Scientific	4448
Microscope coverslips (22x22-1)	Fisher Scientific	12-542-B
M9 Medium			lab made
OP50 bacteria	CGC