Boris Johnson has rejected suggestions of another independence referendum if the UK leaves the EU.
The most high-profile Conservative member of the Leave camp said that Scotland's 2014 vote had been a ‘once in a generation' decision.
"I don't think that's coming around again any time soon,” he added, after a speech in Kent.
The event, in which Mr Johnson also suggested that the UK could secure a Canadian-style free trade deal with the EU, was his first major foray into the referendum campaign since he angered David Cameron by backing a Leave vote.
Just hours earlier Tony Blair had call on pro-EU campaigners to show as much passion in the debate as the London mayor and others.
The former Labour Prime Minister’s remarks were widely interpreted as criticism of his party’s current leader Jeremy Corbyn.
Some Labour MPs are concerned that the veteran socialist is not instinctively pro-EU.
In an attack on politicians like Mr Johnson, and the Ukip leader Nigel Farage, Mr Blair added: “At some point the political class as a whole has got to get up and stand up for itself.
"The centre ground in particular has got to get some more muscularity in its position.
“The ...other major politicians who are fighting this case are no less elitist...
“Framing this argument in the way these guys do: ‘The elites are all for Europe and the people are all against it’ – come on. You guys are just as elitist as anybody else.”
However, Mr Blair played down the idea that he would take a lead role in the In campaign.
Such a move risked “negatives as well as positives”, he admitted.
Meanwhile, a cabinet minister accused the Remain camp of issuing “almost biblical” warnings about the consequences of leaving the EU.
“I listen to these endless comments and speeches about these dire warnings, they’re almost biblical. You expect a plague of frogs and the death of the first born,” Work and Pensions Secretary Mr Duncan Smith said.
He also appeared to criticise his own government’s handling of the economy as he denied a Brexit would create financial uncertainty.
“In December... we were told for the next seven years things were looking great. Within one month of that forecast, we’re now being told that things are difficult ... and therefore we have to revise that," he said.
"I don’t mind that process. All I say to you is if you can’t forecast more than two months, how in heaven’s name can you forecast the next four or five years?”
He was supported by the pro-Leave chairman of the JD Wetherspoon pub chain who accused David Cameron of using “Paisleyite language”.
Tim Martin, who is from Northern Ireland, warned that divisive language was reminiscent of the rhetoric of the former leader of Northern Ireland’s Democratic Unionist party.
For his part Mr Johnson called on voters to ignore the "merchants of gloom" as he said that outside the EU the UK could "take back control of our borders".
The UK could prosper and thrive "as never before" outside the EU, he said, as he suggested the UK could negotiate the kind of deal that Canada has with the EU.
Downing St has for weeks called on those backing a Leave vote to set out what would happen to the UK outside the EU.
But Mr Cameron accused Brexit campaigners of promoting a vision that was "too good to be true".
Meanwhile, another senior Conservative politician announced he is backing a Leave vote, in a fresh blow to the Prime Minister.
London MEP Syed Kamall said he wanted a "fair" points-based immigration system which is "incompatible with membership of the EU".
Elsewhere, the UK's trade deficit with the EU rose to its highest ever monthly total in January, according to newly released figures.
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