Osmolality-controlled fixation and simple preparation of human red blood cells for scanning electron microscopy
Heather N Colvin, Glen Marrs, Regina Joice Cordy
erythrocyte
red blood cell
echinocytosis
scanning electron microscopy
osmolality
microscopy
fixation
human
Abstract
Scanning electron microscopy (SEM) provides a way to visualize red blood cell (RBC) morphology. Previous methods for human RBC fixation can induce osmotic-related changes to healthy RBCs, which can interfere with interpretation of biological morphological changes. In addition, traditional methods for fixation and dehydration of RBCs and associated SEM preparations involve multiple chemicals and time-intensive steps. Here, we provide a simplified protocol for human RBC fixation with careful control of osmolality. This protocol omits the use of sodium cacodylate, osmium tetroxide, and ethanol gradient dehydration steps, yet results in comparable outcomes.
Before start
In order to obtain the human blood needed for these studies, ethical clearance must first be obtained.
Steps
Make osmolality-controlled buffer
-
Add
300µL1X PBS (pH 7.4) +200µLdistilled water (dH2O) in microcentrifuge tube for 500uL total osmolality-controlled buffer solution -
Mix well
NoteOsmolality ≈ 160 mmol/kg Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)
Suspend human RBCs in osmolality-controlled buffer
-
Add
25µLof packed, washed RBCs to500µLosmolality-controlled buffer from Step #1 -
Mix well with pipette, gently
Prepare osmolality-controlled 2% glutaraldehyde solution
- Combine
240µLdH2O,240µL1X PBS (pH 7.4),20µL50% glutaraldehyde in conical tube and mix well by invertingNoteOsmolality ≈ 350 mmol/kg Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)
Fix RBCs with osmolality-controlled glutarldehyde
- Add
500µLosmolality-controlled 2% glutaraldehyde solution from Step #3 slowly to well-mixed RBC suspension from Step #2. - Invert gently in microcentrifuge tube
- Incubate at
Room temperaturein the dark for0h 30m 0sNoteFinal osmolality of fixing solution ≈ 260 mmol/kg Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)
Wash glutaraldehyde-fixed RBCs
-
Centrifuge fixed RBCs after completion of Step #4 at
900x g -
Remove supernatant without disturbing the RBC pellet
Safety informationProperly dispose of supernatant containing glutaraldehyde per institutional guidelines -
Add
1mLdH2O and mix pellet well with pipette -
Centrifuge cells again
900x g -
Remove supernatant without disturbing the RBC pellet
Dilute fixed, washed RBCs in dH2O
-
Mix RBC pellet well with pipette and add
5µLof fixed RBC pellet to995µLdH2O to create at 0.5% hematocrit (hct) suspension of washed and glutaraldehyde-fixed RBCs in water -
Mix well and add
100µL0.5% hct glutaraldehyde-fixed RBC suspension to 12mm round Poly-L-Lysine coated coverslide* on slide warmerNote*Use the most appropriate slide size for the specific scanning electron microscope that will be used to image cells -
Dry slides on slide warmer for
60°C``0h 30m 0s
