Nov 11, 2022

MAP-2 & GFAP Staining Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7y4b1gwz/v1
  • Machlusil Husna1,
  • Kusworini Handono2,
  • Hidayat Sujuti3,
  • Aulanni'am 4,
  • Ettie Rukmigarsari5
  • 1Department of Neurology Faculty of Medicine, Universitas Brawijaya / dr. Saiful Anwar General Hospital, Malang, Indonesia;
  • 2Department of Clinical Pathology Faculty of Medicine, Universitas Brawijaya / dr. Saiful Anwar General Hospital, Malang, Indonesia;
  • 3Department of Ophthalmology Faculty of Medicine, Universitas Brawijaya / dr. Saiful Anwar General Hospital, Malang, Indonesia;
  • 4Department of Chemistry, Faculty of Science Universitas Brawijaya, Malang, Indonesia;
  • 5Faculty of Teacher Training and Education, Islamic University of Malang, Indonesia
Machlusil Husna
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7y4b1gwz/v1
Protocol Citation: Machlusil Husna, Kusworini Handono, Hidayat Sujuti, Aulanni'am , Ettie Rukmigarsari 2022. MAP-2 & GFAP Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y4b1gwz/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2022
Last Modified: November 11, 2022
Protocol  Integer ID: 71479
Keywords: hippocampal excitotoxicity, hippocampal excitotoxicity process, gfap staining protocol neurodegeneration due to neurotoxicity, immediate effect after kainic acid exposure, protocol neurodegeneration due to neurotoxicity, kainic acid exposure, organotypic hippocampal slice culture, neurotoxicity, staining protocol neurodegeneration, gfap staining protocol neurodegeneration, neurodegeneration process, neuronal death, temporal lobe epilepsy, phenomena in temporal lobe epilepsy, astrocyte reactivity, increased calcium
Abstract
Neurodegeneration due to neurotoxicity is one of the phenomena in temporal lobe epilepsy. Experimentally, hippocampal excitotoxicity process can occur due to kainic acid exposure, especially in the CA3 area. Neuronal death, astrocyte reactivity and increased calcium also occur in hippocampal excitotoxicity, but few studies have investigated immediate effect after kainic acid exposure. The organotypic hippocampal slice culture (OHSC) is a useful model for studying the neurodegeneration process, but there are still many protocol differences. In this study, minor modifications were made in the OHSC protocol
Sample Preparation
-Tissue slices were fixed using 4% PFA for 15 minutes
-Wash with PBS for 3 times (5 minutes each)
-Remove PBS and add 0,1% Triton X-100 for 30 minutes
-Wash with PBS for 3 times (5 minutes each)
-Add 10% BSA for 30 minutes at room temperature
Assay Procedure
-Incubate with primary antibody MAP-2 (1:500) and FDAP (1:500) overnight at temperature 4 oC.
-Wash with PBS for 3 times (5 minutes each)
-Incubate with secondary antibody for 1 h at room temperature
- Goat anti-Rabbit IgG-F (1:500) for MAP-2 (Santa Cruz Biotechnology sc-53805, Lot#K1109)
- Anti-mouse Rhodamine (1:500) for GFAP (Rockland, paint : 610-1002)
-Wash with PBS for 3 times (5 minutes each)
-Observe using CLSM with magnification of 100X and a wavelength of 488 nm (MAP-2) and 543 nm (GFAP)