Examples of SEM Coating Fluid on biological samples

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Renal glomerular surface
Kidney-glomerulus-conductive-staining3

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat kidney kidney glomerulus

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat kidney proximal tubule (brush border)
Proximal-tubule-conductive-staining2

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat kidney rlomerular cleavage image (inner blood vessel)
Glomerular-cleavage-conductive-staining2

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat kidney proximal tubule (brush border)
Proximal-tubule-conductive-staining3

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

at liver capillary bile duct
Capillary-bile-duct-conductive-staining2

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat liver rascular endothelial cells
Vascular-endothelial-cells-conductive-staining2

Sample preparation conditions (4 ° C): After fixation with 2% glutaraldehyde perfusion, chopped (thickness approx. 0.5 mm) and re-fixed ⇒ 1% osmium tetroxide fixed 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannin Acid aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 1.5 hour immersion ⇒ Blower, cold air dryer drying Observation Conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat small intestine Microvilli
Microvilli-conductive-staining

Sample preparation conditions (4 ° C): After fixing with 2% glutaraldehyde, shred (thickness approx. 0.5 mm) and re-fix ⇒ Fix with 1% osmium tetroxide 2 hours ⇒ DW wash 10 minutes, 6 times ⇒ 1% tannic acid Aqueous solution 3 hours ⇒ DW washing 10 minutes, 6 times ⇒ 1% osmium tetroxide fixation 2 hours ⇒ DW washing 10 minutes, 3 times ⇒ 2% BEL-1 (70% ethanol) 2 hours immersion ⇒ Blower, cold air dryer drying Observation conditions: SE detector (mixed mix), acceleration voltage 3KV, WD20, emission current (0.8 to 4.7μA)

Rat kidney glomerular cleavage image (inner blood vessel)
Glomerular-cleavage-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Proximal tubule (brush border)
Proximal-tubule-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Kidney glomerulus
Kidney-glomerulus-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Rat kidney proximal tubule (brush border)
ranal-glomerular-surface-conductive-staining2

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Renal glomerular surface
ranal-glomerular-surface-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Rat small intestine soft projection
Soft-projection-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Rat liver capillary bile duct
Capillary-bile-duct-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Rat liver capillary bile duct
Vascular-endothelial-cells-conductive-staining

Double fixing ⇒ 5% BEL-1 (70% ethanol) immersion for 2 hours ⇒ Drying (blower, cold air dryer)

Neutrophil
Neutrophil-conductive-staining

1% GA fixation (slide glass with PLL coating) 30 minutes adhesion (wet) ⇒ DW cleaning ⇒ 10% BEL-1 (diluted solution: 100% ethanol) 10 minutes immersion ⇒ Drying (blower → cold air dryer about 15 minutes) Observation conditions : SE detector Upper emission current 5.5μA

Diatoms
Diatoms-conductive-staining3

2% GA fixed ⇒ DW cleaning ⇒ 10% BEL-1 (70% ethanol) 10 minute immersion ⇒ Drying (blower, cold air dryer) Observation conditions: Acceleration voltage 3KV Emission current: 5.5μA SE detector: Mix

Diatoms
Diatoms-conductive-staining

1% GA ⇒ DW washing ⇒ 10% BEL-1 (70% ethanol) 10 minutes ⇒ Dry (cold air dryer)

Diatoms
Diatoms-conductive-staining2

1% GA ⇒ DW washing ⇒ 10% BEL-1 (70% ethanol) 10 minutes ⇒ Dry (cold air dryer)

Megalopa larva
Megalopa larva-conductive-staining

1% GA ⇒ DW washing ⇒ 10% BEL-1 (70% ethanol) 10 minutes ⇒ Dry (cold air dryer)

Megalopa larva
Megalopa larva-conductive-staining2

1% GA ⇒ DW washing ⇒ 10% BEL-1 (70% ethanol) 10 minutes ⇒ Dry (cold air dryer)

Red blood cells
Red-blood-cells-conductive-staining

10% (diluted solution: 100% ethanol) 10 minutes immersion Acceleration voltage: 3kV Emission current: 5.5μA SE detector: Upper

White blood cells
White-blood-cells-conductive-staining

10% (diluted solution: 100% ethanol) 10 minutes immersion Acceleration voltage: 3kV Emission current: 5.5μA SE detector: Upper

SEM slide glass used
SEM-slide-glass-used-conductive-staining

1% GA fixation (SEM slide glass) 30 minutes adhesion (wet) ⇒ DW cleaning ⇒ 10% BEL-1 (diluted solution: 70% ethanol) 10 minutes immersion ⇒ Drying (blower → cold air dryer about 15 minutes) Observation conditions: SE detector Upper emission current 5.5μA

SEM slide glass used
SEM-slide-glass-used-conductive-staining2

1% GA fixation (SEM slide glass) 30 minutes adhesion (wet) ⇒ DW cleaning ⇒ 10% BEL-1 (diluted solution: 70% ethanol) 10 minutes immersion ⇒ Drying (blower → cold air dryer about 15 minutes) Observation conditions: SE detector Upper emission current 5.5μA

SEM slide glass used
SEM-slide-glass-used-conductive-staining3

1% GA fixation (SEM slide glass) 30 minutes adhesion (wet) ⇒ DW cleaning ⇒ 10% BEL-1 (diluted solution: 70% ethanol) 10 minutes immersion ⇒ Drying (blower → cold air dryer about 15 minutes) Observation conditions: SE detector Upper emission current 5.5μA

SEM slide glass used
SEM-slide-glass-used-conductive-staining4<

1% GA fixation (SEM slide glass) 30 minutes adhesion (wet) ⇒ DW cleaning ⇒ 10% BEL-1 (diluted solution: 70% ethanol) 10 minutes immersion ⇒ Drying (blower → cold air dryer about 15 minutes) Observation conditions: SE detector Upper emission current 5.5μA

Courtesy of St. Marianna University School of Medicine, Electron Microscope Research Facility Chisako Sasaki

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