TITLE: LAMP in situ complete
AUTHORS: Nicholas W. West

[STEPS]
?. [Tissue Fixiation]
Harvest tissue and submerge in an excess of FAA solution, pull a vacuum for ~2 min and agitate samples gently. Hold under vacuum for 2-3 min before slowly releasing vacuum.  Once the vacuum is released the samples should sink, if tissue samples float to the surface, agitate gently and repeat vacuum step if necessary.
?. [Dehydration and Embedding]
2 washes in 50% EtOH, 5 min per wash
?. [Tissue Fixiation]
Incubate samples at   for 10 to 14 hours
4 °C
?. [Dehydration and Embedding]
2 washes in 50% EtOH, 30 min per wash
?. [Dehydration and Embedding]
Incubate 10 min in 75% EtOH at
60 °C
?. [Dehydration and Embedding]
Can be held in 75% EtOH overnight at
4 °C
?. [Dehydration and Embedding]
Incubate 10 min 85% EtOH at
60 °C
?. [Dehydration and Embedding]
Incubate 10 min 95% EtOH at
60 °C
?. [Dehydration and Embedding]
2 washes in 100% EtOH, 10 min per wash at
60 °C
?. [Dehydration and Embedding]
Incubate 10 min 3:1 EtOH (100%) : Histoclear (or xylenes) at
60 °C
?. [Dehydration and Embedding]
Incubate 10 min 1:1 EtOH (100%) : Histoclear (or xylenes) at
60 °C
?. [Dehydration and Embedding]
Incubate 10 min 1:3 EtOH (100%) : Histoclear (or xylenes) at
60 °C
?. [Dehydration and Embedding]
Incubate 5 min Histoclear (or xylenes) at
60 °C
?. [Dehydration and Embedding]
Incubate 10 min 1:1 Histoclear (or xylenes) : Paraffin at
60 °C
?. [Dehydration and Embedding]
5 washes in paraffin at   30 min per wash
60 °C
?. [Dehydration and Embedding]
Can be held overnight in paraffin
?. [Dehydration and Embedding]
Place tissue molds and tweezers/tools in oven (or preffered heating element)
?. [Dehydration and Embedding]
Transfer tissue into pre-warmed tissue molds, fill molds with paraffin and align samples into desired orientation. Wait for bubbles to disperse, accelerate dispersal with gentle agitation if desired. Remove molds from oven and allow to cool
?. [Sectioning]
Tissue sectionsing is a skill and a well angeled and extremely sharp blade is critical. An excellent guide to the technique can be found within "Plant Mircotechnique and Microscopy" by Steven Ruzin 1999
?. [Sectioning]
Use the initial sections to adjust the microtome such that sections ribbon together.  When approaching the perceived area of interest reduce the number of sections per slide. Float section ribbon on   diH2O, capture on slide leaving a wide margin around sample.
45 °C
?. [Sectioning]
Bake tissue sections overnight at ~
45 °C
?. [Dewaxing]
Incubate slides in Histoclear (or xylenes) for 10 min at
55 °C
?. [Dewaxing]
3 washes of Histoclear (or xylenes) at   for 5 min per wash
55 °C
?. [Dewaxing]
3 washes of 100% EtOH at   for 2 min per wash
55 °C
?. [Dewaxing]
Incubate 2 min in 95% EtOH
?. [Dewaxing]
Incubate 2 min in 70% EtOH
?. [Dewaxing]
Incubate 2 min in 50% EtOH
?. [Dewaxing]
Incubate 2 min in DEPC H2O
?. [Dewaxing]
Incubate 2 min in 1xTBS
?. [Dewaxing]
Can be held overnight in 1xTBS
?. [Tissue Prep]
Prepare Proteinase K buffer (1xTE, 0.5% Triton-x-100, 20ug/uL Proteinase K), vortex before adding Proteinase K.
?. [Tissue Prep]
Remove slide, drain excess buffer, and outline tissue with hydrophobic pen/wax pencil
?. [Tissue Prep]
Add   Proteinase K buffer to each slide and place in humidity chamber at   for 20 min
250 µl
37 °C
?. [Tissue Prep]
Wash 3 times with 1xTBS for 5 min per wash
?. [Tissue Prep]
Detecting DNA?  Proceed to LAMP ReactionDetecting RNA?  Remove genomic background first
?. [Tissue Prep]
Can be held overnight at
4 °C
?. [Remove Genomic Background]
Prepare a   DNase I reaction per slide according to manufacturers (Thermo Scientific) instructions
40 µl
?. [Remove Genomic Background]
Prewarm humidity chamber to
37 °C
?. [Remove Genomic Background]
Apply DNase I reaction mix drop-wise onto tissue sections, pitch and roll slide to ensure the reaction covers all tissue samples. Place slide into humidity chamber and incubate at   for 1 hour
37 °C
?. [Remove Genomic Background]
To prevent the reaction mix from pooling and encourage more uniform coverage of tissue samples, every 5-7 min gently pitch and roll the humidity chamber
?. [Remove Genomic Background]
Wash 3 times in 1xTBS, 5 min per wash, proceed to LAMP reaction
?. [Remove Genomic Background]
Can be held overnight at
4 °C
?. [LAMP reaction]
Prewarm humidity chamber to
65 °C
?. [LAMP reaction]
Prepare a   LAMP reaction per slide as outlined in the manufacturers (New England Biosciences) instructions.
30 µl
?. [LAMP reaction]
Apply desired LAMP reaction (colorimeteric or fluorescent) mix drop-wise to the tissue samples, pitch and roll the slide to encourage uniform distribution. Place in humidity chamber and incubate for an empirically determined amount of time, typically between 30 min to 1 hour
?. [LAMP reaction]
To avoid pooling and encourage even distribution of the reaction mix, every 5-7 min gently pitch and roll the humidity chamber for the duration of incubation
?. [LAMP reaction]
2 washes in 1xMAB, 5 min per wash, proceed to detection
?. [Colorimeteric Detection]
Incubate slides in 1xBlocking buffer with gentle rocking at room temperature for 1 hour
?. [Colorimeteric Detection]
Flood slide with   of a 1:5000 anti-DIG alkaline phosphatase conjugated antibody in 1xBlocking buffer, place in humidity chamber and incubate at room temperature for 1 hour
250 µl
?. [Colorimeteric Detection]
Wash 5 times in 1xMAB, 5 min per wash
?. [Colorimeteric Detection]
Flood slides with   NBT/BCIP, incubate at room temperature until reaction develops to satisfactory levels, typically 20-30 min
250 µl
?. [Colorimeteric Detection]
Stop reaction by flooding slides with diH2O, mount a slide cover with your prefered method
?. [LAMP reaction]
Can be held overnight at
4 °C
?. [LAMP reaction]
For colorimeteric detection add   of digoxigenin-11-dUTP, 25nmol (25uL) from Roche, this replaces a portion of the H2O in the reaction mix
0.15 µl
?. [LAMP reaction]
For fluorescent detection use fluorescently labeled loop primer mixture in the standard LAMP reaction
?. [LAMP reaction]
For fluorescent detection procced to step 43