Moreover, our protocol will be less expensive than other methods since the protocol requires fewer neural supplements during neural induction. This article also presents the FM1-43 imaging assay, which is useful for the presynaptic assessment of the iPSCs-derived human neurons. This protocol provides a quick and simplified way to generate NSCs/NPCs and neurons, enabling researchers to establish in vitro cellular models to study brain disease pathology.Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses through immune-electrophysiology in Drosophila melanogaster. This technique allows the quantification of the molecular behavior of the neuromuscular synapses by correlating the structural configuration of the synaptic boutons with their electrical activity.RNA sequencing (RNA-seq) has opened up the possibility of studying virtually any organism at the whole transcriptome level. Nevertheless, the absence of a sequenced and accurately annotated reference genome may be an obstacle for applying this technique to non-model organisms, especially for those with a complex genome. While de novo transcriptome assembly can circumvent this problem, it is often computationally demanding. Furthermore, the transcriptome annotation and Gene Ontology enrichment analysis without an automatized system is often a laborious task. Here we describe step-by-step the pipeline that was used to perform the transcriptome assembly, annotation, and Gene Ontology analysis of Scots pine (Pinus sylvestris), a gymnosperm species with complex genome. Using only free software available for the scientific community and running on a standard personal computer, the pipeline intends to facilitate transcriptomic studies for non-model species, yet being flexible to be used with any organism.Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing analysis. Second, we explain how to perform bioinformatics analysis of sRNA sequence reads using a Galaxy server. In principle, this protocol is suitable to investigate AGO-bound sRNAs from diverse host plants and plant-interacting (micro)organisms.Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA (enzyme-linked immunosorbent assay) and uses electrochemiluminescence to produce measurable signals. Two different antibodies are used in this assay to capture and detect NaV1.1 respectively in brain tissue lysate. The specificity of the antibodies is confirmed by Scn1a gene knock-out tissue. NK-104 calcium The calibration curve standards used in this assay were generated with mouse liver lysate spiked with mouse brain lysate, instead of using a recombinant protein. We showed that this method was qualified and used for quantification of NaV1.1 in mouse brain tissues with specificity, accuracy and precision.Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) configuration. Proteins are overexpressed in E. coli, the cells are transferred into saline solution and the copy number per cell is determined using fluorescence spectroscopy. The suspension is centrifuged and the concentrated cells transferred onto the ATR cell inside the FTIR spectrometer. The thermostatted cell is sealed and illuminated from the top with an LED. Intensity spectra are recorded before and after illumination to generate the difference spectrum of the receptor inside the living cell. With ICIRD spectroscopy, structural changes of soluble photoreceptors are resolved in a near-native environment. The approach works in H2O at ambient conditions, is label free, without any limitations in protein size and does not require any purification step. Graphic abstract In-cell infrared difference spectroscopy on photoreceptors in living E. coli using attenuated total reflection.The ion-selective vibrating probe has been used to detect and quantify the magnitude and direction of transmembrane fluxes of several ions in a wide range of biological systems. Inherently non-invasive, vibrating probes have been essential to access relevant electrophysiological parameters related to apical growth and morphogenesis in pollen tubes, a highly specialized cell where spatiotemporal tuning of ion dynamics is fundamental. Of relevance, crucial processes to the cell physiology of pollen tubes associated with protons and anions have been elucidated using vibrating probes, allowing the identification of diverse molecular players underlying and regulating their extracellular fluxes. The use of Arabidopsis thaliana as a genetic model system posed new challenges given their relatively small dimensions and difficult manipulation in vitro. Here, we describe protocol optimizations that made the use of the ion-selective vibrating probe in Arabidopsis pollen tubes feasible, ensuring consistent and reproducible data.