Protein immunostaining provides important spatio-temporal information about gene expression. Even using high quality antibodies, signal reproducibility and specificity can be problematic depending on tissue fixation methods. For example, formaldehyde fixed tissues often require an epitope retrieval step to expose epitopes of interest for binding to antibodies. One way to achieve this is by using Proteinase K-assisted partial protein degradation (Smith, 1994). However, this process can often reduce, or even abolish immunostaining signals. Here we provide an alternative protocol employing heat induced epitope retrieval (HIER) that gives an improved performance for signal detection (Figure 1).


Figure 1. Comparison of HIER and ProK-assisted epitope retrieval. Immunolocalization images detected using anti-KN1 (A and B), anti-BLH14 antibodies (C and D) and anti-TSH4 (E and F). Tissue sections are subjected to HIER (A, C and E) or Proteinase K (B, D and F)-assisted epitope retrieval. For the Proteinase K treatment, slides are incubated in 1x PBS buffer containing 20 μg/μl of Proteinase K for 30 min at room temperature instead of HIER (Procedure B). Bars are 200 μm.

DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore the original RRBS protocol is not suitable for plants. Accordingly, we developed an in silico pipeline to select specific enzymes to generate a region of interest (ROI)-enriched, e.g., promoter-enriched, reduced representation genome in plants (Hsu et al., 2017). By digesting the maize genome with MseI and selecting 40-300 bp segments, we sequenced about one-fourth of the maize genome while preserving 84.3% of the promoter information. The protocol has been successfully established in maize and can be broadly used in any genome. Our in silico pipeline is combined with the RRBS library preparation protocol, allowing for the computational analysis and experimental validation.

Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the tissue. Here we describe a protocol to utilize micro-computed tomography (micro-CT) scanning to visualize vascular networks in the maize stem. The protocol covers sample fixation and staining with contrasting reagents, data acquisition using micro-CT, reconstructing three-dimensional (3D) models of stem inner structures and extraction of vascular networks from the model. This protocol can be easily applied to various types of species and organs/tissues.

Leaf morphometrics are used frequently by several disciplines, including taxonomists, systematists, developmental biologists, morphologists, agronomists, and plant breeders to name just a few. Leaf shape is highly variable and can be used for identifying species or genotypes, developmental patterning within and among individuals, assessing plant health, and measuring environmental impacts on plant phenotype. Traditional leaf morphometrics requires hand tools and access to specimens, but modern efforts to digitize botanical collections make digital morphometrics a readily accessible and scientifically rigorous option. Here we provide detailed instructions for performing some of the most informative digital geometric morphometric analyses available: generalized Procrustes analysis, elliptical Fourier analysis, and shape features. This comprehensive procedure for leaf shape analysis is comprised of six main sections: A) scanning of material, B) acquiring landmarks, C) analysis of landmark data, D) isolating leaf outlines, E) analysis of leaf outlines, and F) shape features. This protocol provides a detailed reference for applying landmark and outline analysis to leaf shape as well as describing leaf shape features, thus empowering researchers to perform high throughput phenotyping for diverse applications.

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.