2.7.1 Immunocytochemistry
For immunocytochemical analysis of protein expression, cells were plated on 13mm glass coverslips at a density of 5,000 cells per coverslip. Following the required treatment, cells were washed in PBS, fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature and washed again in PBS. Coverslips were stored in PBS at 4°C until use.
For staining, coverslips were quenched with 50 mM ammonium chloride (NH4CL in
PBS) for 10 minutes at room temperature. NH4CL was aspirated and cells were
permeabilised by incubation with 0.2% Triton-X100 for 5 minutes at room temperature, washed 3 times with PBS and blocked for 30 minutes at room temperature with blocking solution (Table 2.3). Primary antibodies were prepared in blocking solution and incubated with the cells for 2 hours at room temperature in a humidified atmosphere with gentle rocking. Primary antibody was aspirated and coverslips were washed 3 times with PBS for 10 minutes per wash. Secondary antibodies were
Compound FinalConcentration Catalogue Number
Lipopolysaccharide (LPS)
1μg/ml for BV2 cells, 100ng/ml for iPSC-MGLC
(in ddH2O)
Sigma #L2880
Mouse macrophage colony
stimulating factor (MCSF) 100ng/ml (in ddH2O) Peprotech #315-02 Adenosine triphosphate
(ATP) 50µM (in ddH2O) Tocris #3245
Mouse complement
component 5a (C5a) 25ng/ml (in ddH2O) R&D Systems #2150-C5 CytochalasinD (CytoD) 10µM (in ddH2O) Santa Cruz Biotechnology #SC201442
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prepared in PBS and incubated with coverslips for 1 hour at room temperature with gentle shaking, followed by 3 further 10 minute washes. Following the final wash, the coverslips were mounted on slides using Vectashield mounting medium with DAPI for fluorescent staining of nuclei. Coverslips were left to dry overnight and were sealed with nail varnish prior to image acquisition on a Zeiss LSM710 confocal microscope.
Table 2.3 Antibodies used for immunocytochemistry
Target Blocking
Solution Primary Antibody Secondary Antibody
TREM2 5% Normal donkey serum Mouse: 10µg/ml sheep anti-TREM2 (AF1729, R&D Systems) Human: 10µg/ml goat anti-TREM2 (AF1828, R&D Systems) Mouse: 1:1000 donkey anti-sheep Alexa Fluor 568 (A-21099, Life Technologies)
Human: 1:500 donkey anti-goat Alexa Fluor 568 (A-11057, Life
Technologies)
Golgi
Complex 3% Bovine Serum Albumin
Mouse: 1:100 rabbit anti- Golgin97 (13192, Cell Signalling Technology) Human: 1:500 mouse anti-Golgin97 (A21270, Life Technologies)
Mouse: 1:1000 goat anti- rabbit Alexa Fluor 488 (R37116, Life
Technologies)
Human: 1:500 goat anti- mouse Alexa Fluor 568 (A11004, Life Technologies) Calnexin 3% Bovine Serum Albumin 1:50 mouse anti-Calnexin (sc-23954, Santa Cruz Biotechnology) 1:500 goat anti-mouse Alexa Fluor 568 (A11004, Life Technologies) Iba1 3% Bovine Serum Albumin 1:500 rabbit anti-Iba1 (019-19741,Wako) 1:1000 goat anti-rabbit Alexa Fluor 488 (R37116, Life Technologies)
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For golgin97 staining, fixation and permeabilisation using methanol was found to result in improved staining. Instead of using 4% PFA and Triton-X100, cells were fixed and permeabilised in ice-cold methanol for 20 minutes at -20°C. The coverslips were washed, blocked in BSA and the protocol continued as for PFA fixation.
2.7.2 Phalloidin labelling of F-actin
Phalloidin staining was performed using Phalloidin CF568 and a standard protocol (Chazotte, 2010), with adjustments made to concentrations and incubation times according to the manufacturer’s instructions (Biotium). Phalloidin, a high affinity actin probe conjugated to a fluorescent dye, was used to study the morphological phenotypes of microglial cells. Cells were plated on 13mm glass coverslips at a density of 5,000 cells per coverslip for BV2 cells. Following treatment with murine macrophage colony-stimulating factor (MCSF, 100ng/ml) or adenosine triphosphate (ATP, 50μM) in serum free RPMI for 5 minutes, cells were washed with PBS and fixed by incubation with 4% PFA for 20 minutes. Cells were washed again and stored in PBS at 4°C until use. Cells were permeabilised with 0.5% Triton-X100 (made up in PBS) at room temperature for 10 minutes. Coverslips were washed 3 times with PBS and incubated with a 1:40 dilution of fluorescent phalloidin at room temperature for 20 minutes, protected from light. Coverslips were washed 3 times with PBS and mounted on slides using Vectashield mounting medium with DAPI for fluorescent staining of nuclei. Coverslips were left to dry overnight and were sealed with nail varnish prior to image acquisition on a Zeiss LSM710 confocal microscope using Zeiss Zen Microscope software.
Cytoskeletal analysis was carried out using ImageJ software (version 1.50). For analysis of filopodia formation, the samples (3 independent experiments, 40-50 cells per sample) underwent blinded measurement of filopodia length and quantification of filopodia number per cell. The samples (5 independent experiments, 40-50 cells per sample) underwent blinded morphological scoring for membrane ruffle formation as: absence, 0, ruffling response 1. Membrane ruffles were defined as wave-like regions of intense F-actin staining at extended cell edges. Immunohistochemistry was performed to confirm that regions counted as ruffles possessed F-actin and Iba1 colocalisation as reported in the literature (Ohsawa et al., 2000).
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2.7.3 Immunohistochemistry
Frozen tissue sections were acquired pre-sliced and mounted from the Queen Square Brain Bank (human temporal lobe samples) and from Amsbio (human kidney samples, Abingdon, United Kingdom) and were stored at -80°C until use. Ethical permission for this study was obtained from the National Hospital for Neurology and Neurosurgery and the Institute of Neurology joint research ethics committee (study reference IONMTA19/16 with corresponding RNA samples acquired under study reference IONMTA8/15). Immunohistochemistry performed using standard protocol with fixation modifications due to the use of fresh frozen tissue (Davies et al., 2013). Slides were fixed and permeabilised immediately upon removal from the freezer with ice cold, 100% methanol for 20 minutes at 4°C, followed by 3 x 5 minute PBS washes. Samples were blocked in 5% horse serum for 1 hour at room temperature, followed by primary antibody incubation (1:250 mouse anti-CR1 SC-7308, Santa Cruz) overnight at 4°C. The slides were washed 3 times with PBS prior to treatment with 0.3% hydrogen peroxide (diluted in PBS) for 15 minutes to block endogenous peroxidases found in the tissue. After further 3 x 5 minute PBS washes, biotinylated secondary antibody was added to the slides (1:200 horse anti-mouse, BA-2001, Vector) for an hour at room temperature. DAB visualisation of antibody staining was performed using Vectastine Elite ABC HRP kit followed by DAB kit according to the manufacturer’s instructions. The slides were dehydrated in serial alcohol dilutions followed by xylene and were mounted in DPX. Images were acquired on a Zeiss Axiophot light microscope.