A Rapid Microwave Mediated Polyethyleneglycol Embedding Method Showing Retention of Intracellular Specialized Metabolites in Leaves of Cinnamomum Tamala (Buch-Ham.) T. Nees & Nees

Polyethyleneglycol (PEG) is considered one of the most effective substitutions for paran in plant histochemistry as an embedding medium. A rapid and straightforward method of PEG embedding has been developed that resulted in a signicant reduction of inltration time than the traditional method of PEG embedding. The material used for PEG embedding was leaves of Cinnamomum tamala, a member of Lauraceae. Samples were put successively in aqueous solutions of PEG 6000 with increasing concentration for inltration. A microwave oven was used as a mode of heating medium. The inltration was completed within 2 h. After the completion of inltration, the samples were embedded in PEG and solidied. Compared with the existing methods available for PEG inltration and embedding, this microwave mediated PEG embedding method saves signicant time; this also saves the tissue from long-term heat-induced damage. Retention of intracellular metabolites, which was not possible in earlier PEG embedded methods, has also demonstrated in the tissue.


Introduction
Polyethyleneglycol (PEG) is one of the most suitable substitutes for para n in animal histochemistry for a long time (Barka and Anderson 1963). The rst reported use of PEG was in 1942 (Richards et al. 1942). PEG can be found in different molecular weights, and along with that, their consistency varies. The earlier use of PEG had been restricted in low molecular weight; one reason for this is the lack of cell walls in animal cells. The use of PEG as an embedding medium was popularized among histochemists only after investigations by Rinehart and Haj (1951). They demonstrated the localization of tissue lipids in samples dehydrated and embedded in PEG. However, for use in plant histochemistry, the high molecular weight PEG was preferred by the researchers (Ferreira et al. 2014(Ferreira et al. , 2017; this may help overcome the cell wall barrier. Very few reports have been found in the use of PEG in plant histochemistry, so it can be deduced that the use of PEG in this eld is not well explored. Researchers have used PEG 6000 in plant histochemistry, which gave a fair and consistent result. Using heat as a form of microwave radiation was started in the eld of histochemistry from the late 1990s (Schichnes et al. 1999 advantage of the microwave method is the reduction in time. In the traditional method, the para n embedding of a plant tissue requires ve to nine days, depending on the hardness (Ruzin 1999). On the other hand, the microwave method brings that down to only ve to seven hours (Schichnes et al. 2001).
This substantial reduction in time helped the researchers to work with the materials more e ciently than earlier.
One of the major advantages of using PEG over para n is that the water-soluble nature of PEG, and thus there is no need for dehydrating the tissue sample (Barka and Anderson 1963). This results in good preservation of internal tissue structure than that of para n embedding methods. As PEG has a higher in ltrating capability than para n, therefore the overall process can be completed within two to three days; this is a major advantage over the para n method as the heat-induced damage of the tissues can be easily avoided ( Samples were transferred in a vial containing a 25% aqueous PEG 6000 solution. The vial containing samples was subjected to kitchen microwave treatment (Panasonic NN-ST266B, 800W) for 5 min at warm mode (~ 40 °C). After decanting the residual 25% PEG solution, the same procedure was repeated for 50% and 90% aqueous PEG solutions. The samples were treated with 90% aqueous PEG solution for two times (5 min each) consecutively, with removal of used and replenishment of fresh 90% PEG. In each case, the volume of the PEG solution was 10 times more of the sample volume. Further utmost care was taken during sample preparation to avoid charring. Embedding was done using 100% aqueous PEG solution followed by storage of blocks at 4 °C until further processing ( Table 1). The traditional PEG embedding method, as described by Ferreira et al. (2014), was also done to compare these two methods.
The blocks were stubbed with a wooden block-holder with the help of molten PEG, as described by

Results And Discussion
Sections obtained from both methods were viewed under bright eld without any staining ( Fig. 1A and B). A few unstained sections were also examined under LED uorescence light (CoolLED pE-3000 White, CoolLED Ltd., UK) tted with Leica DM 2500 LED microscope (Leica, Germany) using 365 nm excitation wavelength and triple-band lter (LED Tripe lter). The sections obtained from the microwave mediated sample showed auto uorescence of the oil present inside the oil cells (Fig. 2B) while such kind of uorescence was not present in traditionally-prepared samples ( Fig. 2A). showed red and pink colours, on the wall of the oil cells respectively, for ruthenium red (Fig. 1E and F) and Schiff reagent (Fig. 1G and H). Sections obtained from both traditional and microwave mediated samples For studying the lipids, sections derived from both the methods were stained with sudan black B for total lipids (Gahan 1984) and with sudan III for neutral lipids (Gahan 1984; Laboratory solution preparation 2020). In both the cases, only oil cells present in the microwave mediated sample sections were found to be stained, showing blackish-blue for sudan black B and orange-red for sudan III (Fig. 2D and F) while the other remain unstained (Fig. 2C and E). Phloroglucinol-HCl was used to stain lignins present in oil cell walls (Laboratory solution preparation 2020), and only sections from microwave-treated sample took light pink colouration (Fig. 2H), which remained absent in the sections obtained by traditional method (Fig. 2G). Nadi staining was done for observation of terpenes (David and Carde 1964), and only the oil cells from the sections of microwave mediated samples showed purple colour (Fig. 2J) while oil cells from the traditionally prepared sections remained unstained (Fig. 2I).
From our study, it has been found that the metabolite retention is only possible in the microwave mediated method. In this method, with the help of different staining we have proposed that our method is better than the traditional PEG embedding method in terms of intracellular metabolite preservation. When stained with toluidine blue O, ruthenium red, Schiff's reagent, and sudan IV, the microwave-based method showed similar results to the traditional PEG embedding method. Bright eld sections (without any staining) also showed a similar type of results as compared to the traditional method. However, when compared with the results obtained from histochemical stainings done with sudan black B, sudan III, phloroglucinol-HCl, and nadi, along with results obtained after excitation under ultraviolet wavelength (at 365 nm), the microwave-based methods show better results than the traditional PEG embedding methods. Intracellular metabolite retention was achieved in microwave-based PEG embedding method as evidenced from the speci c colour produced upon reacting with different stains, which remained absent in the traditional PEG embedding method. Since our work aimed to check the metabolite status at the intracellular level, from that viewpoint, the microwave-based method is more suitable than the traditional method.
The signi cant advantage of this proposed method is its rapidness and can be completed within one hour. Since the process is straightforward and quick, long-term heat-induced damage is less than the classical para n embedding method or traditional PEG embedding method. Another major advantage is the avoidance of tissue dehydration because of the usage of aqueous PEG solutions. As a result, enhanced metabolite retention with reduced cellular distortion can be visualized in the tissue under microscopic examination, which otherwise is impaired upon dehydration processes. Apart from all these advantages, this method is a low cost, since we have used only a kitchen microwave as a mode of heating medium and a simple rotary microtome. The use of the FAA as a xative medium has also made it more affordable, as this is a low cost and most readily available xative. However, there are few limitations that exist with this microwave-mediated PEG embedding method. On a few occasions, microwave-induced heat damage was observed, leading to the secretion of phenolic compounds from the neighboring cells. When compared to other methods of embedding, our method does not show a signi cant improvement in anatomical preservation, and in a few cases, various morphological distortions have also been observed. The last point of di culty we have noted was about the storability of the PEG blocks; these blocks can be stored in 4 C for a good amount of time (up to one month, only). However, once a block is cut, it cannot be stored for further study. Thus, a prepared PEG block once being started cutting, has to be used within 24 hours. This is because since the embedded tissue samples are not dehydrated at the time of preparation, and thus, once cut, the embedded tissue starts to lose water rapidly even at 4 C. Further, these blocks cannot be stored properly at room temperature (25 C).
Nevertheless, this problem is not unique for this new method, as similar problems have been encountered in the traditional PEG embedding method too. This problem can only be overcome by para n embedding method. In our opinion, the less preservation of ner anatomical details should not hamper the overall process, as our goal has been to target the intercellular metabolites, which we achieved with ease by our method. We suggest here that for anatomical observation it is always better to go for classical para n or resin-based embedding methods, as those are well known for obtaining ner anatomical details. The microwave-based PEG embedding method can be used along with traditional para n or resin-based method, which will enable the researcher to study both the anatomical structure as well as the localization of intracellular metabolites in ner details. The embedding procedure reported here is based on our chosen sample i.e., leaf of C. tamala. However, the ner details of the procedure such as in ltration timing, repetition of in ltration dosages may need modi cations depending on the hardness and the type of the tissue sample.

Conclusion
This study showed that the quality of tissue preservation in both traditional PEG embedding method and microwave-based PEG embedding method is identical. Moreover, in microwave-based embedding, the cells retain some of their intracellular metabolite, which is not possible in the traditional method. These encouraging results obtained from this very low-cost and time-saving method can lead to further research on this microwave mediated tissue embedding method and popularize PEG as an embedding medium. From our study, it is evident that this new method can further be utilized for studying a wide range of Author Contributions SS conceived and conducted the microwave embedding experiment, analysed and interpreted the data, and wrote the draft manuscript; RB conducted the traditional embedding experiment and contributed in writing the manuscript too; AM supervised the research and nalized the manuscript. All the authors are in agreement with the results obtained and approved the nal version of the manuscript.

Availability of data and materials
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Competing interests