Immunocytochemistry and CLSM were performed as previously described [74 (link),75 (link)]. Cells were cultured on 12 mm round coverslips coated with poly-D-Lysine/Laminin (BD Bioscience, Franklin Lakes, NJ). Cells were fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were incubated in a blocking buffer containing 1% bovine serum albumin (for MZF1 and SCAND2) or, alternatively, 3% normal goat serum (for SCAND1) in PBS for 30 min, incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at RT for 1 h in the blocking buffer. Cells were washed thrice with PBS for 5 min between the steps. Cells were mounted within ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence images were acquired using Axio Vision CLSM (Zeiss, Oberkochen, Germany) with an AxioCam MR3 (Zeiss) camera for SCAND1, and alternatively, FSX100 inverted microscope (Olympus, Tokyo, Japan) for MZF1 and SCAND2. We used antibodies against MZF1 (C10502, Rb pAb, Assay Biotechnology, Fremont, CA), SCAND1 (ab64828, Rb pAb, Abcam), SCAND2 (5F1, H00054581-M02, Ms mAb, Thermo Fisher Scientific), and anti-rabbit IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific).
Poly d lysine laminin
Manufactured by BD
Sourced in United States
Poly-D-lysine and laminin are coatings used for cell culture substrates. Poly-D-lysine promotes cell attachment, while laminin enhances cell adhesion and growth. These coatings are commonly used to improve the cell culture environment for a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fsx100 inverted microscope, by Olympus (1 mentions)
Axiocam mr3, by Zeiss (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Rb pab, by Abcam (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Hibernate a media, by Transnetyx (1 mentions)
Nbactive4 media, by Transnetyx (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Poly d lysine coated coverslip, by BD (1 mentions)
Collagen 1, by BD (1 mentions)
Fibronectin, by BD (1 mentions)
Poly l lysine (pll), by BD (1 mentions)
6 protocols using poly d lysine laminin
1
Immunocytochemistry and CLSM Analysis
2
Primary Cortical Neuron Cultures Treated with hCG
3
Primary Cortical Neuron Culture Protocol
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Primary cortical neurons obtained from embryonic Day 16 (E16) embryos of a pregnant C57BL/6 mouse or Sprague-Dawley rat were cultured as previously described23 (link). Briefly, cerebral cortices were dissected and incubated with papain solution (HBSS containing 10 U/ml papain, 0.2 mg/ml cysteine, 0.5 mM EDTA, 1 mM CaCl2, and 0.003 N NaOH) for 20 min, followed by the addition of DNase I (Roche, #11284932001). Tissues were dissociated mechanically by pipetting in culture medium and then centrifuged briefly to collect the cells. Cells were plated on poly-D-lysine/laminin-coated plastic dishes or poly-D-lysine/Matrigel-coated (BD Biosciences, San Diego, CA, USA) glass coverslips. The culture medium was changed every 3 days.
4
Immunostaining of Dissociated Ganglion Neurons
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Bilateral NGs resected from mice were immersed in hibernateA media (BrainBits). Each ganglion was cut into five pieces, placed in HibernateA minus Ca2+ media (BrainBits) containing 1 mg/ml collagenase/dispase (Roche Diagnostics) and incubated for 90 min at 37 °C. Neurons were dispersed by gentle titration through pipettes and they were washed three times with fresh NbActive4 media (BrainBits) and plated onto poly-D-lysine/laminin (BD Bioscience)-coated coverslips, followed by incubation (5% CO2 in air at 37 °C) for 24 h. Cultures were rinsed in 10 mM phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB). Ganglion neurons were incubated with the first primary antibodies; anti-GLP-1R or anti-GHSR at 4 °C overnight, and then with the second primary antibodies; anti-GHSR or anti-Pan Neuronal Marker (Supplementary Table S1 ) at 4 °C overnight. They were then reacted with corresponding Alexa Fluor 488- or 594-conjugated secondary IgG (Supplementary Table S1 ) at RT for 1 h. Neurons were observed under a fluorescence microscope (Olympus).
5
Dissociation and Culture of Dorsal Root Ganglia
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Dorsal root ganglia (DRG) were isolated and dissociated according to a previously published method [15 (link)]. Briefly, DRGs were obtained from all spinal locations and dissociated neurons plated on glass coverslips pre-coated with poly-D-lysine/laminin (BD Biosciences) and left to adhere for 1.5–2 hrs before flooding. Growth media consisted of Lebovitz L-15 Glutamax (Life Technologies) supplemented with 10% FCS, 24 mM NaHCO3 and 38 mM glucose.
6
Biofilm Formation on Extracellular Matrices
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For ECM studies, six types of coverslips (5 glass coverslips, each with a different extracellular matrix, and 1 plain glass coverslip) were placed on the bottom of 6 well plates. Collagen I, fibronectin, poly-D-lysine/laminin, poly-L-lysine, poly-D-lysine coated coverslips (BD Bioscience, NJ) and plain glass coverslips were used. 200 ml of A. baumannii inoculum was placed on the coverslips and allowed to sit for 5 min for adherence. Following this, 4 ml of nutrient broth was supplied to all wells and plates and incubated under aerobic conditions for 72 h at 37∞C. Every 12 h during incubation, the medium containing suspended grown bacterial cells was removed and replaced with the same volume of fresh medium. One group was maintained in a dynamic (100 RPM) condition and another in a static condition. After 72 h, biofilms from both the dynamic and static conditions were used for SEM imaging and crystal violet biofilm quantification assay (method as described in the above biofilm yield assay). Prior to SEM imaging, all samples were smoothly rinsed two times using aseptic distilled water. Fixation of biofilms on immersed ECM for crystal violet quantification studies and SEM imaging was performed as per the above de-scribed slanted coverslip method for both the dynamic and static conditions.
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