...Figure 1: Schematic of multiplex immunofluorescence approaches. A) Sequential Multiplex IF, where one target...just want to make some really beautiful images? Multiplex IF may be just what you need! How does it work...work? There are several ways to achieve multiplex IF. One of the most common approaches is to use sequential...before labeling the next target. B) Simultaneous Multiplex IF, where all the primary antibodies are combined...   Figure 2: Examples of simultaneous multiplex IF labeling of rat brain samples from the James...your antibodies When selecting antibodies for multiplexed IF, many of the same considerations apply as ... to avoid secondary antibodies altogether and multiplex with direct IF, using primary antibodies that ...

... Such plasmids are known as multimers, and they are the result of multiple plasmid copies combining in...in a process known as plasmid multimerization. Plasmid Multimerization Early studies on plasmid topology...various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization has ...plasmid multimers function just as well as monomers. However, due to their larger size, multimers may exhibit... detecting plasmid multimers. Digests themselves cannot be used to detect multimers, since restriction...frequency of multimerization, and in some cases completely prevent the formation of plasmid multimers (Bedbrook... pUC multimerization was reduced by growing seed stocks at 30°C compared to 42°C. Screen multiple colonies...

...use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple or ...co-expression of multiple unique genes, you can start with a plasmid that has multiple cloning sites flanking...within the same multicistronic vector, effectively making a construct that expresses multiple unique genes...Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists...promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will create...transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more ... so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work...

...Unless you use a system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from...sites. Modifying multiple genes at once. Using multiplexed gRNAs can target multiple locations in the ...CRISPR makes it easy to target multiple loci - a concept called multiplexing. Since CRISPR is such a robust...you add multiple gRNAs on one plasmid. Sound good? Addgene has many tools to help you multiplex - we’ll...Drosophila, and zebrafish!   Why use multiplexed gRNAs? By expressing multiple gRNAs on the same plasmid, you...clone and transfect multiple promoter-gRNA constructs in order to target multiple sites! In this post,...we'll cover Cas9 multiplexing options, but also check out our blog post about multiplexing with Cpf1.   ...

...simultaneous multi-gene editing with a single crRNA array. Are you interested in using Cpf1 multiplexing in your...for multiplex genome editing.  For a few reasons, Cpf1 is a simplified system for editing multiple targets...protospacer Multiplexing CRISPR-Cas9 options before Cpf1 crRNA array Prior to this new Cpf1 multiplexing method...allow for expression of multiple gRNAs (i.e. Csy4 cleavage sequence, tRNAs, multiple individual promoters...Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method Delivery Method Advantages...Xie et al  Multiplexing crRNA expression with Cpf1  The key advantage of multiplexing crRNA expression... chart below for a summary of multiplexing efficiency in vivo. Multiplex gene editing with CRISPR-Cpf1...

...scalability and efficiency of multiplex gene editing. Moreover, existing multiplex strategies have been facing...developed DAP arrays for efficient multiplex base editing (MBE) and multiplex prime editing (MPE) with only...Schematic of multiplex gene editing with DAP array   Use DAP arrays can enable efficient multiplex gene ...natural CRISPR locus of a bacteria host encodes multiple guide RNAs (gRNAs) on a single array to target...leveraged such host-defense mechanisms to enable multiplex gene editing in a variety of cells and organisms...from a single array, has been a top choice for multiplex gene knockout or regulations, it lacks proper ...contains a double-win for both efficiency and multiplexity, with these constraints? The Gao lab first ...

...Takeshi Imai from Kyushu University. Stochastic multicolor labeling is a powerful solution for discriminating...neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP system to...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced...introducing a mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes...Richi, Marcus N. Leiwe, and Takeshi Imai. "Bright multicolor labeling of neuronal circuits with fluorescent...

...identification. While the Zhang lab controlled their multiplicity of infection so that a single TF was delivered...to each cell, future applications could deliver multiple TFs to each cell, allowing for combinatorial overexpression...dysregulation event. Diseases are often complex, with multiple layers of dysregulation from either genetic mutation...

...will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy...knockout, or multiple gRNAs may be used to target genes with overlapping activity. Multiple gRNAs can also... Addgene Other Techniques for gRNA Multiplexing Multiplexed and programmable regulation of gene networks...;  to observe a phenotype, the modification of multiple genes is often necessary. Guide RNAs (gRNAs) are...editing efficiency. One previous strategy for multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas... test the application of this system to gRNA multiplexing, Xie et al. constructed PTGs containing 8 gRNAs...mutations at all five loci. This highly efficient multiplexing further illustrates the potential of PTGs. Applications...

...biologists (Bohórquez lab), who together developed multifunctional fibers, embedded, and validated simultaneously...adaptable tool that could continuously monitor multiple systems. Ideally this tool would be wireless, ...optical stimulation of its associated fibers across multiple channels and can also record tissue temperature..., Chandrakasan, A., … Anikeeva, P. (2023). Multifunctional microelectronic fibers enable wireless modulation...

...et al. "Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine...

... step in choosing fluorescent proteins for your multi-color imaging experiment is to be aware of what ... multi-color imaging...cell imaging experiments is the ability to follow multiple fluorescently tagged species simultaneously. To...do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation and emission...fluorescent proteins Commonly used filter sets for multicolor imaging include ones designed for CFP, YFP, and...compartments. I hope this sheds some light on multicolor imaging with fluorescent proteins. With the right...

...will highlight the evolution of the  Brainbow multicolor labeling system - a perfect example of the continued...than 3-4 colors due to the tandem integration of multiple copies of the construct (about 8 in Brainbow-1.0...-1.0 mice.) The combinatorial effect of multiple, independent recombination events leads to a rainbow ....1566. Epub 2011 Feb 6. PubMed. Flybow: genetic multicolor cell labeling for neural circuit analysis in ...nmeth.2450. Epub 2013 May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis...13):2835-46. doi: 10.1242/dev.094631. PubMed. Multiplex cell and lineage tracking with combinatorial labels...

...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...Radtke et al., 2020, 2022). I do mean highly multiplexed: you can image over 65 different parameters using...an invaluable resource for anyone performing multiplexed imaging, not just IBEX. The IBEX Imaging Community...., & Germain, R. N. (2020). IBEX: A versatile multiplex optical imaging approach for deep phenotyping ...resources on the Addgene blog Antibodies 101: Multiplex Immunofluorescence Negative Can Be Positive: Open...

...and also makes it hard to compare results across multiple targets because different CAPTURE cell lines will...lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu lab performed...experiment to show that CAPTURE 2.0 could detect multiple chromatin interactions at once. Chromatin interactions...Long-range chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously... a large cluster of enhancers. CAPTURE 2.0’s multiplexing and high resolution allowed the Xu Lab to simultaneously...role in gene regulation The high resolution and multiplexed chromatin ‘photos’ generated with CAPTURE 2.0..., Orkin, S.H., Zhang, M.Q., & Xu, J. (2020). Multiplexed capture of spatial configuration and temporal...

...Stochastic multicolor labeling is a popular technique in neuroscience and developmental biology. This...or entire organism. Because each cell can have multiple copies of the transgene that will recombine independently...persistently fluorescent during development. Stochastic multicolor labeling systems, many based on Brainbow, now... all cell types can be labeled, and that while multiple copies of the transgene can integrate into cells...improving the level of detail possible for stochastic multicolor labeling of the brain. Labeling Drosophila neurons... of transgenic zebrafish lines that allow for multicolor labeling of an entire organism or a specific ...Zebrabow as a system that takes advantage of the multicolor labeling possible from Brainbow. Tracing clonal...

...MoClo application is to assemble multiple unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA...popular DNA assembly tool used to build single- and multi-gene constructs for a variety of applications. MoClo...favorite gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits and ...bacteria MoClo is commonly used to design TUs and multi-TU constructs for synthetic biology applications...assembled into individual TUs or very large (up to 20!) multi-TU constructs (Moore et al., 2016). The variety ... 2016). CIDAR offers a one-pot reaction wherein multi-part assembly can occur. It is quite extensive with...interactions in gene expression, and to generate multi-gene knockouts. Mammalian and fly applications MoClo...

... construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available thanks to ...new tool in Scientific Reports in June. “The multiplexity is one of the most advantageous properties of...established way of making an all-in-one vector for multiplex genome engineering.” Now, they’ve filled that... also be expanded to include even more loci. Multiplex genome engineering demonstrated in human cells...Cas9-nickase vectors work successfully to target multiple sites in cultured human cells. While it has not...be applied to efficient viral vector-mediated multiplex genome engineering. The system now provides an...an efficient targeting strategy for multiplex editing of genomes. It will also allow simultaneous activation...

...can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling of neurons is for...fluorescent neurons. A simple but flawed approach to multi-color labeling is to inject a mix of AAV vectors...less cumbersome than brainbow, another method for multi-color labeling of neurons, because it doesn’t require... system that allows for controlled induction of multi-color labeling of neurons (figure 4). These capsids...lots of individual cells will be transduced with multiple viral particles and labeled a variety of colors...

...Find CROPseq-multi plasmids here Walton, R.T., Qin, Y., Blainey, P.C. (2024). CROPseq-multi: a versatile...developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et ...Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes (iBARs), multiplexed using tRNAs, within...human cells. Addgene offers vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear...articles.  Here's what you'll find in this post: Multiplexed perturbation and decoding in pooled CRISPR screens...System Neurodegeneration Collection updates   Multiplexed perturbation and decoding in pooled CRISPR screens...reduce recombination and avoid separation of the multiplexed elements. The team designed sgRNAs that include...