This video demonstrates a technique for identifying antibodies against specific antigens in a sample using a rat hippocampal neuronal culture. By exposing cultured neurons to patient samples containing autoantibodies targeting N-Methyl-D-aspartate receptors (NMDARs), the presence of autoantibodies bound to the neuronal surface is revealed through immunostaining.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. PART 1: Establishment of dissociated, fetal, rodent hippocampal neuronal cultures 1. Preparative steps Poly-L-Lysine (PLL) coating of the plating surfaces (3 days before dissection) Prepare the borate buffer by adding 2.38 g of boric acid and 1.27 g of borax to 500 mL of distilled water and st…
Representative Results
Figure 1: Visual protocol for primary cultures of hippocampal neurons. (A) Flowchart showing the three parts of the protocol for preparing dissociated-cell cultures of hippocampal neurons from embryonic rats at E18. The protocol is divided into 1. Hippocampal isolation, 2. Cell dissociation, and 3. Cell seeding. (B) Selection of recommended tools for hippocampal …
Declarações
The authors have nothing to disclose.
Materials
10 cm Cell culture dish
Nunc
12-565-020
12 mm round coverslips
Fisher
NC9708845
20x NA 0.75 S Fluor air objective
Nikon
CFI Super Fluor 20X
3.5 cm Cell culture dish
Nunc
12-565-90
6 cm Cell culture dish
Nunc
12-565-94
B27 supplement
Gibco
17504-044
Beaker 100 mL
Pirex
–
Borax
Sigma-Aldrich
B9876
Boric Acid
Sigma-Aldrich
B0252
Curved forceps
FST
11009-13
D-Glucose
Sigma-Aldrich
D9434
DMEM High Glucose (4.5 g/L), without L-Glutamine, without Phenol Red
Capricorn
DMEM-HXRXA
Female Wistar rat (18-days pregnant)
Janvier
–
Fetal Bovine Serum (FBS)
Biowest
S181B-500
Fine-angled forceps
FST
11251-35
Fine-curved forceps
FST
11272-30
Fine-straight forceps
FST
11251-23
FITC filter cube
Nikon
Standard Series
Forceps
FST
11000-12
Goat anti-Human AF488
Invitrogen
A11013
HBSS
Capricorn
HBSS-1A
HEPES
Sigma-Aldrich
H3375
Hibernate-E medium
Gibco
A12476-01
Horse Serum (HS)
Thermofisher
26050088
Human anti-NMDAR antibody (CSF)
Patient Sample
–
Human anti-NMDAR antibody (Serum)
Patient Sample
–
ImageJ/Fiji
NIH
v1.50i
Inverted fluorescence microscope
Nikon
Eclipse TE2000-U
Phosphate-Buffered Saline
Gibco
10010023
Poly-L-Lysine (PLL)
Peptide international
OKK-35056
Polystyrene ice tray
–
–
Precision spring-scissors
FST
15000-08
ProLong Gold with DAPI (antifading mounting media)