TITLE: Preparation of human Red Blood Cells for Scanning Electron Microscopy imaging
AUTHORS: Marie Anne Balanant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SEM imaging can be used to quantify RBC shape. RBCs are sampled from fresh blood samples or from packed RBC units during storage. RBCs are resuspended into PBS to make up 500 μL of solution at 5% haematocrit. RBCs are then fixed by progressively adding a concentrated 2% glutaraldehyde solution in the cell suspension. The cells are incubated for 30 min at RT and in the dark, before being centrifuged and washed in PBS. This fixation protocol was established in order to limit RBC shape changes due to the presence of glutaraldehyde.</div><div class = "text-block">After fixation, a RBC suspension at 2.5% haematocrit is adhered to coverslips coated with poly-D-lysine. A second fixation step is realised on the adhered RBCs by incubating the coverslips in osmium tetroxide in cacodylate buffer (1%) for one hour. The coverslips are then dried by incubating them in an ascending series of ethanol, then by incubating them with hexamethyldisilizane (HMDS) for 30 min. The HMDS incubation step is repeated twice before the coverslips are dried in open air. Finally, the coverslips are gold coated and imaged with a Zeiss Sigma FESEM.</div></div>

[STEPS]
?. [Coverslip preparation]
Prepare glass coverslips by incubating them in  poly-D-lysine solution in PBS for  at RT
10 Volume Percent
?. [Coverslip preparation]
Rinse twice with milliQ water
?. [Coverslip preparation]
Dry and keep at  until use
?. [RBC fixation with glutaraldehyde]
Wash  of RBC in  PBS ()
100 µl
900 µl
[at 1000g]
?. [RBC fixation with glutaraldehyde]
Resuspend  of the RBC suspension in  of PBS
25 µl
475 µl
?. [RBC fixation with glutaraldehyde]
Add  of  glutaraldehyde solution in PBS to the cell suspension slowly (drop by drop) to reach a final glutaraldehyde concentration of 1%
500 µl
2 Volume Percent
Work under a fume hood when using glutaraldehyde
?. [RBC fixation with glutaraldehyde]
Incubate for  in the dark
?. [RBC fixation with glutaraldehyde]
Wash with  of PBS ()
1 ml
[at 1000g]
?. [Adhesion on coverslip]
Dilute  of fixed cells suspension into  PBS
100 µl
400 µl
?. [Adhesion on coverslip]
Incubate  of diluted cell suspension on a poly-D-lysine coated coverslip () in corresponding buffer (in 6-wells plates)
200 µl
?. [Adhesion on coverslip]
Rince slide 2 times with PBS (without scratching/damaging cell layer)
?. [SEM sample preparation]
Incubate in  cacodylate buffer () . Repeat step 3 times
0 Molarity (M)
Work under a fume hood during this section, check waste disposal for the different chemicals
?. [SEM sample preparation]
Post-fix with  osmium tetroxide ()
1 Mass Percent
?. [SEM sample preparation]
Rinse twice with milliQ water ()
?. [SEM sample preparation]
Dehydrate the sample with ethanol- Ethanol  ()- Ethanol  ()- Ethanol  ()- Ethanol  ()- Ethanol  ()- Ethanol ()- Ethanol  ()- Ethanol  ()
40 Volume Percent
50 Volume Percent
60 Volume Percent
70 Volume Percent
80 Volume Percent
90 Volume Percent
100 Volume Percent
100 Volume Percent
?. [SEM sample preparation]
Incubate in HDMS (). Repear step twice
?. [SEM sample preparation]
Dry under the hood for  minimum (place filter paper under coverslip to prevent attaching to the culture plate)
?. [SEM sample preparation]
Cover with a layer of gold palladium