TITLE: Phenotypic analysis of root nodules based on paraffin fixation
AUTHORS: Carolina Cervera Torres, Kalpana Nanjareddy, Manoj-Kumar Arthikala

[DESCRIPTION]
The mutualistic relationship between leguminous plants and rhizobia bacteria stands out as one of the most crucial, primarily because of its capacity to convert atmospheric nitrogen (N2) into ammonia (NH3). Recent years have witnessed a surge in studies focusing on the regulation and establishment of this symbiosis, leading to the development of protocols aimed at scrutinizing the phenotype of the pivotal structures facilitating nutrient exchange between the symbiotic partners – the nodules. The subsequent protocol outlines a highly effective method for paraffin processing of nodules, enabling a comprehensive phenotypic analysis. This protocol serves as a valuable analytical tool for research endeavors delving into the intricate details of the rhizobia-legume symbiosis.

[STEPS]
SECTION: Sample collection
1. Precision in sample collection is crucial to prevent any damage. Submerge the roots in water to facilitate the handling of nodules. Utilizing tweezers and delicate scissors, carefully sever the roots approximately 1-2 cm away from the nodule. This ensures ease of manipulation during processing without disrupting the nodule anatomy.

SECTION: Fixation
2. Immerse the tissues in 5 mL of FAA fixative solution (10% formalin, 5% glacial acetic acid, 50% ethanol, and 35% ddH2O) or Carnoy's solution (60% ethanol, 30% chloroform, 10% glacial acetic acid, and 1 gof ferric chloride).

SECTION: Fixation
3. Place the tissues in the fixative within a vacuum chamber for  20 minutes, breaking the vacuum at 10-minute intervals.
 
    

SECTION: Paraffin embedding
4. Following fixation, immerse the samples in 5 mL of a 1X PBS solution for 10 minutes on ice.

SECTION: Paraffin embedding
5. Dehydrate the samples in a 5 mL ethanol series at room temperature: 30% ethanol for 30 minutes, 50% ethanol for 30 minutes, 70% ethanol for 1 hour, 85% ethanol for 30 minutes, 95% ethanol for 30 minutes, and 100% ethanol twice for 30 minutes each.
 


SECTION: Paraffin embedding
6. Infiltrate the samples with 5 mL of ethanol and xylene substitute (Histochoice) for 30 minutes at room temperature, using the following concentrations: ethanol/xylene substitute (2:1, v/v), ethanol/xylene substitute (1:1, v/v), ethanol/xylene substitute (1:2, v/v), and pure xylene substitute twice.

SECTION: Paraffin embedding
7. Infiltrate the samples with 5 mL of xylene and paraffin substitute in glass bottles overnight, mixing in an oven at 60 °C. Use the following concentrations: xylene/paraffin substitute (2:1, v/v), xylene/paraffin substitute (1:1, v/v), xylene/paraffin substitute (1:2, v/v).

SECTION: Paraffin embedding
8. Continue to make 4 changes of paraffin infiltration of tissues with 5 ml of pure paraffin with an interval of 5-8 hrs and incubate in a hot air oven at 60 °C.

SECTION: Preparation of blocks and cuts
9. Pour approximately 15 mL of paraffin into a plastic Petri dish, and carefully empty the tissues from the jars.

SECTION: Preparation of blocks and cuts
10. Use tweezers to manipulate and arrange the nodules in the desired orientation within the Petri dish. If additional tissue coverage is required, add a small amount of paraffin. Allow the blocks to dry for 24 hours.
SECTION: Preparation of blocks and cuts
11. Prepare the slides to be used a day before making cuts by immersing them in a 2% gelatin solution.
SECTION: Preparation of blocks and cuts
12. Mount the paraffin blocks containing tissue onto wooden pegs. Attach these pegs to the microtome for sectioning at the desired thickness.
SECTION: Preparation of blocks and cuts
13. Place the cut sections onto gelatin-coated slides and position them on a hot plate (50-60 °C) until the tissue spreads out evenly. Discard any excess gelatin from the slides.

SECTION: Preparation of blocks and cuts
14. Leave the slides to dry until the next day.
 


SECTION: Histochemical staining
15. The slides were deparaffinized in 150 mL of xylene substitute (histochoice) for 15 minutes, gently shaken every 5 minutes. The slides were deparaffinized in 150 mLof xylene substitute (histochoice) for 15 minutes, gently shaken every 5 minutes.

SECTION: Histochemical staining
16. Subsequently, the samples were rehydrated using solutions of varying ethanol concentrations (100%, 95%, 70%, 50%, and 30%) in ddH2O with each step lasting 3 minutes at room temperature.
SECTION: Histochemical staining
17. Following rehydration, the samples were placed in a container with ddH2O for an additional 3 minutes.
SECTION: Histochemical staining
18. For staining, a 0.1% Toluidine blue solution in ddH2O was prepared.
SECTION: Histochemical staining
19. Samples were immersed in the staining solution for 20 seconds, followed by three rinses in ddH2O, each lasting 10 seconds.
SECTION: Histochemical staining
20. Dehydration was carried out by briefly treating the samples in 95% ethanol and 100% ethanol for 10 seconds at room temperature.
SECTION: Histochemical staining
21. The final step involved immersing the samples in xylene substitute for 5 minutes. 
 

SECTION: Histochemical staining
22. Approximately 400 µL of xylene-based mounting medium was applied to the slide, and the cover glass was carefully lowered, avoiding the formation of any air bubbles that might interfere with obtaining high-quality images.

SECTION: Histochemical staining
23. The slides were left to air dry overnight before observation and photography at the desired magnifications.