Jose Miguel Rojas , did you try embedding into resins? Instead of paraffin you may use hard resins such a epon hard, durcupan, araldite or LR White hard. They are used for electron microscopy but when you cut semithin sections (500-1000um), you can observe them in light microscope as well.

The sample need to be properly infiltrated with resin.

Lenka

Jose Miguel Rojas , did you try embedding into resins? Instead of paraffin you may use hard resins such a epon hard, durcupan, araldite or LR White hard. They are used for electron microscopy but when you cut semithin sections (500-1000um), you can observe them in light microscope as well.

The sample need to be properly infiltrated with resin.

Lenka

I agree that resin would provide better support but it depends on if there are any further protocols that you may want to perform on the slides after sectioning.

For paraffin it may held to soak the blocks in water, if you trim to the seed in the block and then place it into water for twenty minutes the seed tissues will absorb the water and soften a little, then cool the block as much as possible to make the wax as hard as possible before putting back to the blade.

If the paraffin is breaking, there are two possibilities

(1) the paraffin is hard that is why it is breaking. Check the melting point of your paraffin. The choice of paraffin to be used depend on your climate which determine your ambient temperature. For example I live in Nigeria a tropical climate, I found that paraffin with 56 to 60 degree centigrade produced better ribbon.

(2) your knife may be blunt, another minor possibility is your clearance angle, that is the position of your knife to the specimen.

Hi there,

the breaking/peeling off the paraffin often results from difference in hardness of embedded material and embedding medium and/or from heterogeneity of material itself. If the first is the problem, you can try softening the material prior to embedding. For wood/bark anatomy storing in ethylenediamine 4%-10% solution for a few days prior to embedding in paraffin proved to be useful (sometimes I store wood in the solution and cut it on a sledge microtome without embedding and it works fine). Probably the same method can be used for seeds. For further reference see:

1. Carlquist S. 1982. The use of ethylenediamine in softening hard plant structures for paraffin sectioning. Stain Technology 57: 311–317.

However, if the latter is the problem and sample is heterogenous, meaning it consists of softer and harder parts, it'll be more reasonable to follow one of the protocols specifically developed to face such a problem. Since seeds are notoriously difficult to cut, there're plenty of them. You can try e.g.,

1. Barbosa ACF, Pace MR, Witovisk L, Angyalossy V. 2010. A new method to obtain good anatomical slides of heterogenous plant parts. IAWA Journal 31: 373–383.

2. Benedict JC. 2015. A new technique to prepare hard fruits and seeds for anatomical studies. Applications in Plant Sciences 3: 1500075.

3. Ferreira BG, Teixeira CT, Isaias RMS. 2014. Efficiency of the Polyethylene-Glycol (PEG) Embedding Medium for Plant Histochemistry. Journal of Histochemistry and Cytochemistry 62: 577–583.

4. Ribeiro RC, Oliveira DMT. 2015. Small and hard seeds: a practical and inexpensive method to improve embedding techniques for light microscopy. Acta Botanica Brasilica 28: 624–630.

There's also an article I like, though only for historic reasons, in which the authors used variety of household detergents and multitude of materials (incl. such of animal origin), so I'm adding it more as a curiosity:

1. Pearlman RC, Cole BC. 1951. Softening of hard tissue for sectioning. Biotechnic and Histochemistry26: 115–118.

Good luck with your section!

Kamil